Nr3b nmda receptor subunit compositions and related methods

ABSTRACT

The invention relates to the identification of a new mouse gene and its trancript that encode a new NR3B subunit of NMDA receptors.

RELATED APPLICATIONS

This application claims the benefit under 35 USC 119 of U.S. provisionalapplication Ser. No. 60/344,545, filed Oct. 19, 2001, now pending.

FIELD OF THE INVENTION

The present invention relates to novel NR3B genes and transcripts thathave been cloned from mouse. The invention is directed to the isolatedNR3B nucleic acids, the polypeptides encoded by these nucleic acids,agents that selectively bind thereto, and various diagnostic,therapeutic, and research uses of these compositions.

BACKGROUND OF THE INVENTION

Glutamate is a major excitatory neurotransmitter in the mammaliancentral nervous system. Its action is mediated by two distinct classesof glutamate receptors, ionotropic and metabotropic glutamate receptors(Seeburg, 1993; Nakanishi et al., 1996; Dingledine et al., 1999). Theionotropic glutamate receptor is a ligand-gated cation channel thatpasses cation upon glutamate binding. It consists of several differentsubclasses including NMDA, AMPA, kainate, and δ, which, in turn, arehetero-oligomers of several subunits (Seeburg, 1993; Hollmann andHeinemann, 1994; Dingledine et al., 1999). The NMDA receptor is ahetero-oligomer of NR1, NR2A, NR2B, NR2C, NR2D, and NR3A (also calledχ-1 or NMDAR-L) subunits. NR1 is a key subunit that confers essentialfunctions of the NMDA receptor and is expressed ubiquitously in thecentral nervous system. In contrast, other subunits show more limitedexpression and confer a functional diversity. For example, duringdevelopment of the visual cortex, NR2B is expressed predominantly in theearly stages. After functional maturation of the cortex, predominantexpression switches to NR2A (Quinlan et al., 1999). Concomitantly, thedecay time course of the NMDA receptor-mediated synaptic current becomesfaster (Carmignoto and Vicini, 1992; Philpot et al., 2001). Dark rearingof animals, which prolongs the critical period of the visual cortex,delays this conversion (Quinlan et al., 1999).

In contrast with NR2 subunits, less is known about the NR3 class ofsubunit. NR3A is expressed ubiquitously during development and itsexpression level reaches a maximum at around the first postnatal week.Thereafter, the level gradually decreases, and in adult animals, NR3A isconfined to limited nuclei in the thalamus, amygdala, and nucleus of thelateral olfactory tract (Ciabarra et al., 1995; Sucher et al., 1995).The NR3A subunit binds to NR1 and NR2 and acts in a dominant-negativefashion against the NMDA receptor to reduce whole-cell current as wellas single-channel conductance (Ciabarra et al., 1995; Sucher et al.,1995; Das et al., 1998; Perez-Otano et al., 2001). Consistent with thisrole for NR3A, mice lacking NR3A have a larger NMDA receptor-mediatedcurrent and an increased dendritic spine density in cerebrocorticalneurons (Das et al., 1998), suggesting that the NR3A subunit plays arole in the development and plasticity of the central nervous systemthrough a modulation of NMDA receptor function.

There are a number of degenerative motor neuron disorders that may belinked to aberrant NMDA receptor function. One such disorder isamyotrophic lateral sclerosis (ALS). In this progressive motor neurondisorder, motor neurons consisting of anterior horn cells of the spinalcord and upper or corticospinal motor neurons die, which results in aninexorable loss of muscle innervation and atrophy, eventually leading todeath of the patient. Mutations in superoxide dismutase (SOD) have beensuggested as one of the causes of ALS, but may only account for a verysmall population of patients (˜2%) with the disease.

The diagnosis of motor neuron diseases such as ALS is difficult andthere is no available treatment to eliminate or reverse the inexorableloss of motor neuron function in this disease. Although a transgenic SODanimal is available as a model for the limited SOD-based ALS, good cellmodels or animal models for many motor neuron diseases such asnon-SOD-based ALS are not available. If such models were available, theycould foster research into the cause and physiological progression ofmotor neuron diseases. In addition, motor neuron disease models areneeded for screening possible candidate molecules for use as much neededmedications for the disease. Motor neuron disease models would also beuseful for developing and testing treatment methods and could helpincrease the likelihood of positive outcome for patients with motorneuron disease such as ALS.

SUMMARY OF THE INVENTION

Novel NR3B genes and transcripts that may be utilized to providemuch-needed information about motor neuron diseases, including ALS, haveidentified and cloned from mouse. These novel sequences and thepolypeptides they encode are useful for developing and utilizingdiagnostic methods and therapeutic methods and for producing animalmodels for research into prevention and treatment of motor neurondisease. The lack of suitable animal models for many motor neurondiseases, including a model applicable for examination of most ALSetiologies, prevents better understanding of the parameters of thediseases. The identification of the novel mouse NR3B nucleic acids andpolypeptides they encode can be used to generate cell and animal modelsof diseases, including but not limited to ALS, which are useful forcharacterizing motor neuron diseases.

According to one aspect of the invention, isolated nucleic acidmolecules are provided. The isolated nucleic acid molecules are selectedfrom the group consisting of (a) nucleic acid molecules which hybridizeunder high stringency conditions to a nucleic acid molecule having anucleotide sequence set forth as SEQ ID NO:1, providing that no morethan about 18% of the nucleotides are changed from SEQ ID NO:1, (b)nucleic acid molecules that differ from the nucleic acid molecules of(a) in codon sequence due to the degeneracy of the genetic code, and (c)complements of (a) or (b), wherein the nucleic acid molecules orcomplements thereof code for a mouse NR3B NMDA receptor subunit.

In some embodiments, the isolated nucleic acid molecule comprises anucleic acid sequence set forth as: SEQ ID NO: 1.

According to another aspect of the invention, isolated nucleic acidmolecule selected from the group consisting of: (a) fragment ofnucleotides 1-3290 of SEQ ID NO: 1 between 24 and 3289 nucleotides inlength, providing that no more than about 18% of the nucleotides arechanged from SEQ ID NO:1, and (b) complements of (a) are provided.

According to another aspect of the invention expression vectors areprovided. The expression vectors include the-foregoing isolated nucleicacid molecules operably linked to a promoter.

According to yet another aspect of the invention, host cells with theforegoing expression vectors are provided.

According to still another aspect of the invention, transgenic non-humananimals that include the aforementioned expression vectors are provided.

According to another aspect of the invention, isolated polypeptidesencoded by the aforementioned isolated nucleic acid molecule areprovided. In some embodiments, the isolated polypeptide includes anamino acid sequence selected from the group consisting of SEQ ID NO: 2and a fragment or functional variant of SEQ ID NO:2.

According to another aspect of the invention, binding polypeptides thatselectively bind the aforementioned isolated polypeptide are provided,and the binding polypeptide is an antibody or antigen-binding fragmentthereof. In some embodiments, the binding polypeptide selectively bindsthe polypeptide sequence of SEQ ID NO: 2.

According to yet another aspect of the invention, compositions theinclude a molecule selected from the group consisting of: (a) thenucleic acid of any of claims 1-3, (b) the polypeptide encoded by theisolated nucleic acid molecule of any of claims 1-3, and (c) the bindingpolypeptide of any of claims 9-10, and a pharmaceutically acceptablecarrier are provided.

According to another aspect of the invention, methods for makingmedicaments are provided. The methods include placing in apharmaceutically acceptable carrier, a molecule selected from the groupconsisting of: (a) the isolated nucleic acid molecules of any of claims1-3, (b) the isolated polypeptide of any of claims 7-8, and (c) thebinding polypeptides of any of claims 9-10. Some embodiments of themethods also include the step of placing comprises placing atherapeutically effective amount of the molecule selected from the groupin the pharmaceutically acceptable carrier to form one or more doses.

According to another aspect of the invention methods of making aglutamate receptor in vitro are provided. The methods includeintroducing glutamate receptor nucleic acids into a cell, wherein theglutamate receptor nucleic acids encode a NR3B polypeptide. In someembodiments of the method the NR3B subunit is encoded by the nucleotidesequence set forth as SEQ ID NO: 1.

According to another aspect of the invention, methods for diagnosing amotor neuron disorder characterized by aberrant expression of a NR3Bmolecule are provided. The -methods include: detecting expression of aNR3B molecule in a first biological sample obtained from a subject,wherein a difference in expression level of the NR3B molecule comparedto expression level a NR3B molecule in a control sample indicates thatthe subject has a motor neuron disorder characterized by aberrantexpression of a NR3B molecule. In some embodiments, the methods alsoinclude detecting expression of a NR3B molecule in a second biologicalsample obtained from the subject at a time subsequent to the firstbiological sample, and comparing the expression of the NR3B molecule inthe first biological sample and the second biological sample as anindication of the onset, progression, or regression of the motor neurondisorder.

In some embodiments, a decrease in expression level of the NR3B in thesecond biological sample compared to the expression level in the NR3B inthe first biological sample indicates progression of the motor neurondisorder characterized by aberrant expression of NR3B. In otherembodiments, an increase in expression level of the NR3B in the secondbiological sample compared to the expression level in the NR3B in thefirst biological sample indicates regression of the motor neurondisorder characterized by aberrant expression of NR3B. In preferredembodiments, the motor neuron disorder is amyotrophic lateral sclerosis(ALS). In some embodiments, the motor neuron disorder characterized byaberrant expression of a NR3B molecule is amyotrophic lateral sclerosis(ALS). In certain embodiments, the methods include detecting expressionof a NR3B nucleic acid molecule. In some embodiments, the NR3B nucleicacid molecule comprises the nucleic acid sequence set forth as SEQ IDNO: 1 or fragment thereof. In certain embodiments, the methods includedetecting expression of a NR3B polypeptide. In certain embodiments, theNR3B polypeptide is set forth as SEQ ID NO: 2 or fragment thereof. Insome embodiments, detecting comprises contacting the biological samplewith an agent that selectively binds the NR3B molecule. In someembodiments, the NR3B molecule is a nucleic acid and wherein the agentthat selectively binds the NR3B molecule is a nucleic acid selected fromthe group of nucleic acid molecules comprising the nucleotide sequencesthat hybridize to SEQ ID NO: 1 under high stringency conditions. Incertain embodiments, the NR3B molecule is a polypeptide and wherein theagent that selectively binds the NR3B molecule is a binding polypeptideselected from the group of binding polypeptides that selectively bind toSEQ ID NO: 2. In some embodiments, the biological sample is selectedfrom the group consisting of: a neuronal cell, neuronal tissue, andspinal fluid.

According to another aspect of the invention, methods for evaluating theeffect of candidate pharmacological compounds on expression of an NR3Bsubunit of a glutamate receptor are provided. The methods includeadministering a candidate pharmaceutical agent to a subject; determiningthe effect of the candidate pharmaceutical agent on the expression levelof NR3B relative to the expression level of NR3B in a subject to whichno candidate pharmaceutical agent is administered, wherein a relativeincrease or relative decrease in the expression level of NR3B indicatesthe effect of the candidate pharmaceutical compound on the expression ofthe NR3B subunit of the glutamate receptor. In preferred embodiments,the subject is a mouse. In preferred embodiments, the glutamate receptoris an NMDA receptor.

According to yet another aspect of the invention, methods for evaluatingthe effect of candidate pharmacological compounds on the expression of aNR3B subunit of a glutamate receptor are provided. The methods includecontacting a candidate pharmaceutical agent with a NR3B subunitexpressing cell or tissue sample; determining the effect of thecandidate pharmaceutical agent on the expression level of NR3B relativeto the expression level of NR3B in a NR3B subunit expressing cell ortissue sample not contacted with the candidate pharmaceutical agent,wherein a relative increase or relative decrease in the expression levelof NR3B indicates the effect of the candidate pharmaceutical compound onthe expression of NR3B subunit of a glutamate receptor. In someembodiments, the glutamate receptor sample is in culture. In preferredembodiments, the glutamate receptor sample is an NMDA receptor sample.

According to another aspect of the invention, methods for diagnosing amotor neuron disorder characterized by aberrant function of a NR3Bmolecule are provided. The methods include detecting function of a NR3Bmolecule in a first biological sample obtained from a subject, wherein adifference in function of the NR3B molecule compared to a NR3B moleculein a control sample indicates that the subject has a motor neurondisorder characterized by aberrant function of a NR3B molecule. Someembodiments also include: detecting function of a NR3B molecule in asecond biological sample obtained from the subject at a time subsequentto the first biological sample, and comparing the function of the NR3Bmolecule in the first biological sample and the second biological sampleas an indication of the onset, progression, or regression of the motorneuron disorder. In some embodiments, a decrease in function level ofthe NR3B in the second biological sample compared to the function levelin the NR3B in the first biological sample indicates progression of themotor neuron disorder characterized by aberrant function of NR3B. Inpreferred embodiments, the motor neuron disorder is amyotrophic lateralsclerosis (ALS). In certain embodiments, an increase in function levelof the NR3B in the second biological sample compared to the functionlevel in the NR3B in the first biological sample indicates regression ofthe motor neuron disorder characterized by aberrant expression of NR3B.

In some embodiments, the motor neuron disorder characterized by aberrantfunction of a NR3B molecule is amyotrophic lateral sclerosis (ALS). Someembodiments include comprising detecting function of a NR3B nucleic acidmolecule. In certain embodiments, the NR3B nucleic acid moleculecomprises a nucleic acid sequence set forth as SEQ ID NO: 1 or fragmentthereof. Some embodiments include detecting function of a NR3Bpolypeptide. In certain embodiments, the NR3B polypeptide is set forthas SEQ ID NO: 2 or fragment thereof. In some embodiments, detectingincludes determining the cation passage through an NMDA receptorchannel. In certain embodiments, the cation flux is determined with amethod selected from the group consisting of: electrophysiologicalrecording, drug screening assays, and ion-flux measurement. In someembodiments, the biological sample is selected from the group consistingof: a neuronal cell, neuronal tissue, and spinal fluid.

According to still another aspect of the invention, methods forevaluating the effect of candidate pharmacological compounds on functionof an NR3B subunit of a glutamate receptor are provided. The methodsinclude administering a candidate pharmaceutical agent to a subject thatexpresses a glutamate receptor containing a functional NR3B subunit;detecting the function of the NR3B subunit of the glutamate receptor,determining the effect of the candidate pharmaceutical agent on thefunction level of NR3B relative to the function level of NR3B in asubject to which no candidate pharmaceutical agent is administered,wherein a relative increase or relative decrease in the function levelof NR3B indicates the effect of the candidate pharmacological compoundon the function of the NR3B subunit of the glutamate receptor. Inpreferred embodiments, the subject is mouse. In preferred embodiments,the glutamate receptor is an NMDA receptor. In some embodiments,detecting includes determining the cation passage through an NMDAreceptor channel. In certain embodiments, the cation flux is determinedwith a method selected from the group consisting of:electrophysiological recording, drug screening assays, and ion-fluxmeasurement.

According to another aspect of the invention, methods for evaluating theeffect of candidate pharmacological compounds on function of a NR3Bsubunit of a glutamate receptor are provided. The methods includecontacting a glutamate receptor sample with a candidate pharmaceuticalagent; detecting the function of the NR3B subunit of the glutamatereceptor, determining the effect of the candidate pharmaceutical agenton the function level of NR3B relative to the function level of NR3B ina glutamate receptor sample not contacted with the candidatepharmaceutical agent, wherein a relative increase or relative decreasein the function level of NR3B indicates the effect of the candidatepharmacological agent on the function of NR3B subunit of the glutamatereceptors. In some embodiments, the glutamate receptor sample is inculture. In some embodiments, the glutamate receptor sample is an NMDAreceptor sample. In certain embodiments, detecting includes determiningthe cation passage through an NMDA receptor channel. In someembodiments, the cation flux is determined with a method selected fromthe group consisting of: electrophysiological recording, drug screeningassays, and ion-flux measurement.

According to yet another aspect of the invention, kits for diagnosing amotor neuron disorder associated with aberrant expression of a NR3Bmolecule are provided. The kits include one or more nucleic acidmolecules that hybridize to a NR3B nucleic acid molecule under highstringency conditions and instructions for the use of the nucleic acidmolecules in the diagnosis of a motor neuron disorder associated withaberrant expression of a NR3B molecule. In some embodiments, the one ormore nucleic acid molecules are a first primer and a second primer and,wherein the first primer and the second primer are constructed andarranged to selectively amplify at least a portion of an isolated NR3Bnucleic acid molecule comprising SEQ ID NO: 1.

According to another aspect of the invention, kits for diagnosing aNR3B-associated motor neuron disorder in a subject are provided. Thekits include one or more binding polypeptides that selectively bind to aNR3B polypeptide, and instructions for the use of the bindingpolypeptides in the diagnosis of a motor neuron disorder associated withaberrant expression of a NR3B molecule. In some embodiments, the one ormore binding polypeptides are antibodies or antigen-binding fragmentsthereof. In certain embodiments, the NR3B polypeptide is encoded by anucleic acid comprising a nucleotide sequence set forth as SEQ ID NO:1.

According to another aspect of the invention, methods for treating asubject with a motor neuron disorder characterized by decreasedexpression of a NR3B molecule are provided. The methods includeadministering to the subject an amount of a NR3B nucleic acid moleculeeffective to increase expression of a NR3B polypeptide and treat themotor neuron disorder.

According to yet another aspect of the invention, methods for treating asubject with a motor neuron disorder characterized by decreasedexpression of a NR3B polypeptide are provided. The methodsinclude-administering to the subject an amount of a NR3B polypeptideeffective to treat the motor neuron disorder.

According to yet another aspect of the invention, methods for treating asubject with a motor neuron disorder characterized by increasedexpression of a NR3B nucleic acid molecule are provided. The methodsinclude administering to the subject an amount of an antisense moleculeto a NR3B nucleic acid molecule effective to treat the motor neurondisorder.

According to yet another aspect of the invention, methods for treating asubject with a motor neuron disorder characterized by increasedexpression of a NR3B polypeptide are provided. The methods includeadministering to the subject an amount of a NR3B polypeptide bindingpolypeptide effective to treat the motor neuron disorder. In someembodiments, the binding polypeptide agent is an antibody or anantigen-binding fragment thereof

According to another aspect of the invention, methods for treating asubject with a motor neuron disorder characterized by decreased functionof a NR3B molecule are provided. The methods include administering tothe subject an amount of a NR3B nucleic acid molecule effective to treatthe motor neuron disorder.

According to another aspect of the invention, methods for treating asubject with a motor neuron disorder characterized by decreased functionof a NR3B polypeptide are provided. The methods include administering tothe subject an amount of a NR3B polypeptide effective to treat the motorneuron disorder.

According to yet another aspect of the invention, methods for treating asubject with a motor neuron disorder characterized by increased functionof a NR3B nucleic acid molecule are provided. The methods includeadministering to the subject an amount of an antisense molecule to aNR3B nucleic acid molecule effective to treat the motor neuron disorder.

According to still another aspect of the invention, methods for treatinga subject with a motor neuron disorder characterized by increasedfunction of a NR3B polypeptide are provided. The methods includeadministering to the subject an amount of a NR3B polypeptide bindingpolypeptide effective to treat the motor neuron disorder.. In someembodiments, the binding polypeptide agent is an antibody or anantigen-binding fragment thereof.

According to another aspect of the invention, methods for producing aNR3B polypeptide or fragment-thereof are provided. The methods includeproviding an isolated NR3B nucleic acid molecule operably linked to apromoter, wherein the NR3B nucleic acid molecule encodes the NR3Bpolypeptide or fragment thereof, and expressing the NR3B nucleic acidmolecule in an expression system. In some embodiments, the methods alsoinclude isolating the NR3B polypeptide or a fragment thereof from theexpression system. In some embodiments, the NR3B nucleic acid moleculeis set forth as SEQ ID NO: 1.

According to another aspect of the invention, methods for making a NR3Bpolypeptide are provided. The methods include culturing the host cell ofclaim 5, and isolating the NR3B polypeptide from the culture.

According to another aspect of the invention, methods for preparing amodel of a motor neuron disease characterized by aberrant expression ofa NR3B molecule are provided. The methods include introducing into acell, a NR3B molecule. In some embodiments, the motor neuron disorder isAmyotrophic Lateral Sclerosis (ALS). In some embodiments, the NR3Bmolecule is a NR3B nucleic acid molecule set forth in SEQ ID NO: 1. Incertain embodiments, the NR3B molecule is a NR3B polypeptide set forthin SEQ ID NO: 2. In some embodiments, the cell is in a non-human animalsubject. In some embodiments, the model is a knock-out model.

According to another aspect of the invention, methods for preparing ananimal model of a motor neuron disorder characterized by aberrantfunction of a NR3B molecule are provided. The methods includeintroducing into a non-human subject, an aberrant NR3B molecule; anddetecting expression of the aberrant NR3B molecule in a first biologicalsample obtained from the non-human subject. In some embodiments, theaberrant NR3B molecule is not functional. In certain embodiments, theaberrant NR3B molecule has increased function level compared to acontrol NR3B function level. In some embodiments, the aberrant NR3Bmolecule has decreased function level compared to a control NR3Bfunction level. In preferred embodiments, the motor neuron disorder isAmyotrophic Lateral Sclerosis (ALS). In certain embodiments, the NR3Bmolecule is a NR3B nucleic acid molecule. In some embodiments, the NR3Bmolecule is a NR3B polypeptide.

According to another aspect of the invention, methods for preparing anon-human animal model of a motor neuron disorder characterized byreduced expression of a NR3B molecule are provided. The methods includeadministering to a non-human subject an effective amount of ananti-sense molecule to a NR3B nucleic acid molecule to reduce expressionof the NR3B nucleic molecule in the non-human subject. In someembodiments, the NR3B molecule is a nucleic acid molecule selected fromthe group containing SEQ ID NO: 1 and SEQ ID NO:3.

According to yet another aspect of the invention, methods for preparinga non-human animal model of a motor neuron disorder characterized byreduced expression of a NR3B molecule are provided. The methods includeadministering to a non-human subject an effective amount of a bindingpolypeptide to a NR3B polypeptide to reduce expression of the NR3Bpolypeptide in the non-human subject. In some embodiments, the bindingpolypeptide agent is an antibody or an antigen-binding fragment thereof.In certain embodiments, the NR3B molecule a nucleic acid moleculeselected from the group containing SEQ ID NO: 1 and SEQ ID NO:3.

All of the foregoing aspects of the invention relate to the motor neurondisorders and diseases described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The descriptions include drawings that may reference color components.The figures are illustrative only and are not required for enablement ofthe invention disclosed herein.

FIGS. 1A-1D show digital images relating the structure of the glutamatereceptor subtype NR3B. (A) Phylogenic tree of the members of theglutamate receptor family based on the neighbor joining method. Thehorizontal length of the branch indicates the distance betweenpolypeptides. (B) Mouse and human NR3B sequences (SEQ ID NO:3 and SEQ IDNO:4 amino acids 1-890). The human sequence is not complete at theC-terminus. Identical amino acids between two species are indicated by“:” and homologous ones by ”.”. SP and M1˜M4 indicate the predictedsignal peptide and membrane-associated region, respectively. Putativeglycosylation sites are boxed. Upward arrowheads indicate exonboundaries. Amino acids implicated in ligand binding of GluR2 from acrystallographic study are shown below the mouse sequence at thecorresponding positions. (C) A comparison of the M2 domain that formschannel pore (GluR1, GluR2, GluR5, KA-1, δ2, NR1, NR2A, NR3A, NR3B areSEQ ID NOs:15-23 respectively). The critical amino acid at the Q/R/Nsite, which controls ion permeability and rectification, is glycinefollowed by an arginine in both NR3B and NR3A (shown under arrow). Inhuman NR3B, the Q/R/N site is an arginine (B). (D) Genomic structure ofmouse NR3B. Open and shaded boxes are coding and non-coding regions,respectively. The 5′-non-coding region may extend further upstream.

FIG. 2A-2D contain digital photomicrographic images depicting tissuedistribution of mouse NR3B. FIG. 2A is a gel demonstrating NR3Btranscripts detected by RT-PCR with primers that span exons 1 and 2. ThePCR product of the expected length was detected in the brainstem (BS)and spinal cord (Sp) and, to a much lesser extent, in the cerebellum(Cb). When reverse transcriptase was omitted, (-RT), the product was notdetected. As a positive control for RT-PCR, the NR1 was amplified inparallel, producing two bands that correspond to different splicevariants. OB, olfactory bulb; Cx, cerebral cortex; Str, striatum; Hip,hippocampus; Th-MB, thalamus to midbrain region. FIG. 2B depictsNorthern analysis of mouse spinal cord polyA+RNA, which detected asingle band at around 3.5 kb. FIG. 2C shows In situ hybridization ofsagittal sections of mouse brain with ³³P-labeled antisense (AS) andsense (S) probes. The antisense probe detected a discrete signal intrigeminal motor (V), facial (VIII), and ambiguous nuclei (IX). Thecerebellum also had a faint signal. FIG. 2D shows higher magnificationof cranial nerve nuclei. A restricted expression of NR3B was detected inmotor neurons that control somatic movement but not in those controllingocular movement. Left: low and high magnification images ofNissl-stained sections of oculomotor (III), trochlear (IV), trigeminalmotor (V), facial (VII) and abducens (VI) nuclei are shown. Right: Insitu hybridization using anti-sense and sense probes labeled withdigoxigenin. Strong signal was observed in trigeminal motor and facialnuclei but not of those innervating extraocular muscles. Bars, 1 mm forC and 100 μm for D.

FIG. 3A, 3B shows digitized photomicrographic images of expression ofNR3B in spinal cord. Left panel: low-magnification images of sectionsstained with Nissl and adjacent sections hybridized with antisenseprobe. Right panel: higher magnification of the same sections. Somaticmotor neurons are strongly labeled in C2 and L2 levels. In contrast, theexpression of NR3B was significantly weaker in the motor neurons at theL6 level. Surrounding these cells, there was a plexus of serotoninimmunoreactive fibers (arrow heads), which is one of characteristics ofmotor neurons in Onuf's nucleus. Bars, 100 μm.

FIG. 4A-4D contain digitized images of glutamate-induced whole-cellcurrent recorded from HEK293 cells expressing NR3B. NR3B was coexpressedwith the NR1 and NR2A subunits. FIG. 4A shows sampleelectrophysiological traces of glutamate-induced current recorded at −80mV to +60 mV (20 mV step) in the presence or absence of 1 mM Mg²⁺. FIG.4B shows the remaining current in NR3B-expressing cells exhibits a Mg²⁺block indistinguishable from cells not expressing NR3B. FIG. 4C showsNR3B3 acts as a dominant-negative subunit and suppressesglutamate-induced whole-cell current in cells coexpressing NR1 and NR2A.The distribution of averaged responses obtained at +60 mV in Mg²⁺-freesolution is shown in a cumulative plot. Increasing the amount of NR3Bplasmid (0, 1, and 3 μg) versus other subunits (each 1 μg) caused aconcomitant decrease in current amplitude. AMPA receptor mediatedcurrent was unchanged by the coexpression with NR3B. FIG. 4D illustratesthe coexpression with NR3B did not change the expression levels of NR1or NR2A. NR1 and NR2A tagged with GFP were individually expressed withNR3B and their expression levels were measured by the fluorescenceintensity. The statistical significance in this figure was assessed byKolmogorov-Smirnov test.

DETAILED DESCRIPTION OF THE INVENTION

The present invention, in one aspect, involves the cloning of CDNAs thatencode a mouse and human NR3B polypeptide. The sequence of the codingportion of the mouse gene is presented as SEQ ID NO: 1, and thepredicted amino acid sequence of this gene's polypeptide product ispresented as SEQ ID NO: 2. The sequence of the coding portion of thehuman gene is presented as SEQ ID NO: 3, and the predicted amino acidsequence of this gene's polypeptide product is presented as SEQ ID NO:4.

Sequence analysis revealed that the mouse NR3B polypeptide shows 51%similarity to the NR3A subunit of the NMDA receptor. The invention thusinvolves in one aspect mouse NR3B polypeptides, nucleic acid moleculesencoding those polypeptides, functional modifications and variants ofthe foregoing, useful fragments of the foregoing, as well as therapeuticand diagnostic products (including antibodies), non-human animal models,and methods relating thereto.

As used herein, the term “NR3B” means mouse NR3B and the term “hNR3B”means human NR3B. As used herein, the term “NR3B polypeptide” refers topolypeptides/proteins with a deduced structure with the same generalcharacteristics as a glutamate receptor subunit with an N-terminalsignal peptide and four membrane-associated regions. Five (in mouse) orfour (in human) consensus sequences for N-glycosylation sites are foundon the N-terminal domain and one (for both species) at the loop betweenthe third and fourth membrane-associated regions. The intracellularC-terminus of mouse NR3B has three threonines and five serines, whichmay serve as regulatory phosphorylation sites. The C-terminus ends withAla-Glu-Ser, which does not conform to the consensus PDZ domainprotein-binding site sequence typical of other glutamate receptor familymembers (Songyang et al., 1997; Sheng and Sala, 2001).

As used herein, a subject is a human, non-human primate, cow, horse,pig, sheep, goat, dog, cat, or rodent, including but not limited to:guinea pig, rat, and mouse. As used herein an animal is a non-humanprimate, cow, horse, pig, sheep, goat, dog, cat, or rodent, including,but not limited to: guinea pig, rat, and mouse. In all embodiments,human and mouse NR3B molecules and human and mouse subjects arepreferred.

As used herein, the term “NR3B-associated motor neuron disorder” meansany motor neuron disorder or motor neuron disease characterized byaberrant expression of NR3B. Motor neuron diseases and disorders mayinclude, but are not limited to: Amyotrophic lateral sclerosis (ALS):hereditary ALS, also called Lou Gehrig's disease or Maladie de Charcot(e.g.: dominant, recessive, neurofilament heavy subunit: 22q12,superoxide dismutase: 21q22.1, childhood-onset ALS, ALS2, ALS4, ALS5);ALS with bulbar onset; benign course and bunina bodies; SporadicAmyotrophic Lateral Sclerosis; Primary Lateral Sclerosis; PrimaryMuscular Atrophy (PMA); Western Pacific ALS; Western Pacific ALS-likedisorders; Insulinoma; Monomelic Amyotrophy; Bulbar Syndromes (e.g.Brown-Vialetto-van Laere, Fazio-Londe, Congenital Bulbar Syndrome,Kennedy's Syndrome, Madras motor neuron disease, Spino-bulbar MuscularAtrophy, Worster-Drought syndrome, Congenital Suprabulbar Paralysis);Sporadic Bulbo-spinal Muscular Atrophy; Motor Neuropathy, (e.g. distal:GM1 or GalNAc-GDla antibody and multifocal (MMN): GM1 antibody);myopathies (e.g. Paraneoplastic Motor Neuropathy; Paraneoplastic LowerMotor Neuron syndrome; Poliomyelitis and Post-polio syndrome; DiabeticAmyotrophy; Acute Axonal Motor Neuropathy, Porphyria); Spinal MuscularAtrophy (SMA) [e.g. hereditary, SMN (5q), androgen receptor, bulbar SMA,distal, hexosaminidase A (Tay-Sachs), HMN 1, H 2, HMN, HMN 5B, HMN 7(vocal cord), HMN J, ulnar-median, diaphragmatic paralysis and neonatal,SPG 14, upper limb predominance, dominant, proximal SMA, benigncongenital with contractures, congenital with leg weakness, MSN-P(Okinawa type), Scapuloperoneal syndromes, Congenital withArthrogryposis, Werdnig-Hoffman, Kugelberg-Welander, Spinal MuscularAtrophy 2 (SMA2), X-linked Infant SMA and artirogryposis, and toxic];Primary Lateral Sclerosis2; Atypical Motor Neuron diseases withophthalnoplegia and extrapyramidal disorders4; multiple systems atrophy,sporadic or autosomal dominant; striatonigral degeneration; Adult-onsetsporadic olivopontocerebellar atrophy (OPCA); Shy-Drager syndrome;Polyglucosan Body disease; motor neuronopathy with cataracts andskeletal abnormalities; Disinhibition-dementia-Parkinsonism-amyotrophycomplex (DDPAC); neuropathies (e.g. myopathies: distal; MyotonicDystrophy; Inclusion body myositis; Myasthenia Gravis; peripheral nervelesion; median: recurrent motor branch, anterior interosseus, ulnar:guyon canal, radial: posterior interosseus, brachial plexopathy, bulbarinvolvement, Bulbo-spinal Muscular Atrophy, androgen receptor: X-linked;toxic: (e.g. lead; dapsone; botulism; tick paralysis); infections (e.g.Polio, Central European encephalitis, Creutzfeld-Jacob); Amyotrophy;Polyneuropathy (±Demyelinating); Multifocal Motor Neuropathy; (MMN);Hopkins' Syndrome (e.g. acute post-asthmatic amyotrophy); Hirayama'sdisease; O'Sullivan-McLeod syndrome (e.g. slow progression); Gowers;Machado-Joseph; and Arthrogryposis-lower motor neuron disease.

As used herein, the term “aberrant” refers to decreased expression(including zero expression) or increased expression of the natural NR3Bmolecule (nucleic acid or polypeptide) as compared to its expression ina subject who does not have a NR3B-associated disorder. Aberrantexpression can also refer to increased expression of a mutant NR3Bmolecule (nucleic acid or polypeptide) as compared to its expression ina subject who does not have a NR3B-associated disorder. For example, theaberrant expression is expression that is not about 100% of the level ofNR3B in a subject free of a NR3B-associated disorder. In anotherexample, the level of NR3B expression could be outside of the range ofexpected levels in normal subjects. Aberrant expression may bedetermined by comparing levels of NR3B molecules to those levels incontrols. The control(s) include positive and negative controls whichmay be a predetermined value that can take a variety of forms. Thecontrol(s) can be a single cut-off value, such as a median or mean, orcan be established based upon comparative groups, such as in groupshaving normal amounts of NR3B and groups having abnormal amounts of NR3Bmolecules.

As used herein, a compound or signal that “modulates the activity of anNMDA receptor” refers to a compound or signal that alters the activityof NMDA receptors so that activity of the NMDA receptor is different inthe presence of the compound or signal than in the absence of thecompound or signal. In particular, such compounds or signals includeagonists and antagonists. The term agonist refers to a substance orsignal, such as NMDA, that activates receptor function; and the termantagonist refers to a substance that interferes with receptor function.Typically, the effect of an antagonist is observed as a blocking ofactivation by an agonist. Antagonists include competitive andnon-competitive antagonists. A competitive antagonist (or competitiveblocker) interacts with or near the site specific for the agonist (e.g.,ligand or neurotransmitter). A non-competitive antagonist or blockerinactivates the functioning of the receptor by interacting with a siteother than the site that interacts with the agonist.

As understood by those of skill in the art, assay methods foridentifying compounds that modulate NMDA receptor activity (e.g.,agonists and antagonists) generally require comparison to a control. Onetype of a “control” cell or “control” culture is a cell or culture thatis treated substantially the same as the cell or culture exposed to thetest compound, except the control culture is not exposed to testcompound. For example, in methods that use voltage clampelectrophysiological procedures, the same cell can be tested in thepresence and absence of test compound, by merely changing the externalsolution bathing the cell. Another type of “control” cell or “control”culture may be a cell or a culture of cells which is identical to thetransfected cells, except the cells employed for the control culture donot express functional NR3B subunits. In this situation, the response oftest cell to test compound is compared to the response (or lack ofresponse) of receptor-negative (control) cell to test compound, whencells or cultures of each type of cell are exposed to substantially thesame reaction conditions in the presence of compound being assayed.

Another example of a comparative group is a group having a particulardisease, condition and/or symptoms and a group without the disease,condition and/or symptoms.

Another comparative group is a group with a family history of aparticular disease and a group without such a family history of theparticular disease. The predetermined control value can be arranged, forexample, where a tested population is divided into groups, such as alow-risk group, a medium-risk group and a high-risk group or intoquadrants or quintiles, the lowest quadrant or quintile beingindividuals with the lowest risk or highest expression levels of NR3Band the highest quadrant or quintile being individuals with the highestrisk or lowest expression levels of NR3B molecules.

The predetermined value of a control will depend upon the particularpopulation selected. For example, an apparently healthy population willhave a different “normal” NR3B expression level range than will apopulation which is known to have a condition characterized by aberrantNR3B expression. Accordingly, the predetermined value selected may takeinto account the category in which an individual falls. Appropriateranges and categories can be selected with no more than routineexperimentation by those of ordinary skill in the art. By “abnormallyhigh” it is meant high relative to a selected control. Typically thecontrol will be based on apparently healthy individuals in anappropriate age bracket or based on normally expressed NMDA receptors.

It will also be understood that the controls according to the inventionmay be, in addition to predetermined values, samples of materials testedin parallel with the experimental materials. Examples include samplesfrom control populations or control samples generated throughmanufacture to be tested in parallel with the experimental samples.

According to one aspect of the invention, an isolated nucleic acidmolecule is provided. The isolated nucleic acid molecule is selectedfrom the group consisting of: (a) nucleic acid molecules which hybridizeunder high stringency conditions to a nucleic acid molecule having anucleotide sequence set forth as SEQ ID NO: 1 and that code for a NR3Bpolypeptide, (b) nucleic acid molecules that differ from the nucleicacid molecules of (a) in codon sequence due to the degeneracy of thegenetic code, and (c) complements of (a), no more than about 18% of thenucleotides in (a) are changed from SEQ ID NO: 1, and the sequencesexclude nucleic acids having nucleotide sequences that are containedwithin SEQ ID NO: 1 and that are known as of the filing date of thisapplication. Preferably, no more than about 17%, 16%, 15%, 14%, 13%,12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the nucleotidesare changed relative to SEQ ID NO:1 in the nucleic acid molecules thathybridize under stringent conditions to SEQ ID NO:1.

The preferred isolated nucleic acids of the invention are NR3B nucleicacid molecules which encode a NR3B polypeptide. As used herein, a NR3Bpolypeptide refers to a polypeptide that is encoded by a nucleic acidincluding the nucleotide sequence set forth as SEQ ID NO: 1, or afunctional fragment thereof, or a finctional equivalent thereof (e.g., anucleic acid sequence encoding the same polypeptide as encoded by SEQ IDNO: 1), provided that the finctional fragment or equivalent encodes apolypeptide which exhibits a NR3B functional activity. As used herein, aNR3B functional activity refers to the ability of a NR3B polypeptide tomodulate one or more of the following parameters: NMDA receptoractivity, cation passage, and/or NMDA ligand binding. Although notwishing to be bound to any particular theory or mechanism, it isbelieved that the NR3B polypeptide may affect at least some of theabove-noted cell functions by participating in the varied subunitcontrol of glutamate receptors such as the NMDA receptor in human andanimal cells. NR3B functional activity can be determined, for example,by measuring the ability of NMDA receptors to pass cations uponactivation with ligand. Such measurement can be performed byelectrophysiological recording coupled with application of ligand,agonists, antagonists, and control chemicals, using proceduresunderstood by those of skill in the art. (See, for example, theelectrophysiological assay described in the Examples).

In preferred embodiments, the isolated NR3B nucleic acid molecule is SEQID NO: 1 and the isolated NR3B polypeptide is SEQ ID NO: 2.

The invention provides isolated nucleic acid molecules which code forNR3B polypeptides and which hybridize under high stringency conditionsto a nucleic acid molecule consisting of the nucleotide set forth in SEQID NO: 1. Such nucleic acids may be DNA, RNA, composed of mixeddeoxyribonucleotides and ribonucleotides, or may incorporate syntheticnon-natural nucleotides. Preferably this group of sequences excludeshNR3B sequences. Various methods for determining the expression of anucleic acid and/or a polypeptide in cells are known to those of skillin the art and are described further below and in the Examples. As usedherein, the term polypeptide is meant to include large molecular weightproteins and polypeptides and low molecular weight polypeptides orfragments thereof.

The term “high stringency conditions” as used herein refers toparameters with which the art is familiar. Nucleic acid hybridizationparameters may be found in references which compile such methods, e.g.Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds.,Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989, or Current Protocols in Molecular Biology, F. M. Ausubel, etal., eds., John Wiley & Sons, Inc., New York. More specifically, highstringency conditions, as used herein, refers, for example, tohybridization at 65° C. in hybridization buffer (3.5×SSC, 0.02% Ficoll,0.02% polyvinyl pyrrolidone, 0.02% Bovine Serum Albumin (BSA), 2.5 mMNaH₂PO₄ (pH 7), 0.5% SDS, 2 mM EDTA). SSC is 0.15M sodiumchloride/0.015M sodium citrate, pH 7; SDS is sodium dodecyl sulphate;and EDTA is ethylenediaminetetracetic acid. After hybridization, themembrane upon which the DNA is transferred is washed at 2×SSC at roomtemperature, and then at 0.1×SSC/0.1×SDS at temperatures up to 68° C.

The foregoing set of hybridization conditions is but one example of highstringency hybridization conditions known to one of ordinary skill inthe art. There are other conditions, reagents, and so forth which can beused, which result in a high stringency hybridization. The skilledartisan will be familiar with such conditions, and thus they are notgiven here. It will be understood, however, that the skilled artisanwill be able to manipulate the conditions in a manner to permit theclear identification of homologs and alleles of NR3B nucleic acidmolecules of the invention. The skilled artisan also is familiar withthe methodology for screening cells and libraries for expression of suchmolecules which then are routinely isolated, followed by isolation ofthe pertinent nucleic acid molecule and sequencing.

In general homologs and alleles typically will share at least 80%nucleotide identity and/or at least 80% amino acid identity to SEQ IDNO: 1 and SEQ ID NO: 2, respectively, in some instances will share atleast 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99, 99.5% nucleotide identity and/or atleast 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99, 99.5% amino acid identity. The percentidentity can be calculated using various publicly available softwaretools developed by NCBI (Bethesda, Md.) that can be obtained through theinternet (ftp:/ncbi.nlm.nih.gov/pub/). Exemplary tools include the BLASTsystem available at http://www.ncbi.nlm.nih.gov, which uses algorithmsdeveloped by Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997).Pairwise and ClustalW alignments (BLOSUM30 matrix setting) as well asKyte-Doolittle hydropathic analysis can be obtained using the MacVectorsequence analysis software (Oxford Molecular Group). Watson-Crickcomplements of the foregoing nucleic acid molecules also are embraced bythe invention.

In screening for NR3B genes, a Southern blot may be performed using theforegoing conditions, together with a detectably labeled probe (e.g.radioactive or chemiluminescent probes). After washing the membrane towhich the DNA is finally transferred, the membrane can be placed againstX-ray film or a phosphorimager to detect the radioactive orchemiluminescent signal. In screening for the expression of NR3B RNA,Northern blot hybridizations using the foregoing conditions can beperformed on samples taken from subjects suspected of having a conditioncharacterized by aberrant expression of a NR3B molecule, e.g., abnormalNMDA receptor function and/or abnormal NR3B polypeptide expression.Amplification protocols such as PCR using primers that hybridize to thesequences presented also can be used for detection of the NR3B genes orexpression thereof

Identification of related sequences can be achieved using PCR and otheramplification techniques suitable for cloning related nucleic acidsequences. Preferably, PCR primers are selected to amplify portions of anucleic acid sequence believed to be conserved (e.g., a channel poreconformation domain, a ligand binding domain, etc.). Again, nucleicacids are preferably amplified from a tissue-specific library (e.g.,neuronal tissue such as spinal cord and brain). One-also can useexpression cloning-utilizing the antisera described herein-to identifynucleic acids that encode related proteins.

The invention also includes degenerate nucleic acid molecules whichinclude alternative codons to those present in the native materials. Forexample, serine residues are encoded by the codons TCA, AGT, TCC, TCG,TCT, and AGC. Each of the six codons is equivalent for the purposes ofencoding a serine residue. Thus, it will be apparent to one of ordinaryskill in the art that any of the serine-encoding nucleotide triplets maybe employed to direct the protein synthesis apparatus, in vitro or invivo, to incorporate a serine residue into an elongating NR3B protein.Similarly, nucleotide sequence triplets that encode other amino acidresidues include, but are not limited to: CCA, CCC, CCG, and CCT(proline codons); CGA, CGC, CGG, CGT, AGA and AGG (arginine codons);ACA, ACC, ACG and ACT (threonine codons); AAC and AAT (asparaginecodons); and ATA, ATC and ATT (isoleucine codons). Other amino acidresidues may be encoded similarly by multiple nucleotide sequences.Thus, the invention embraces degenerate nucleic acids that differ fromthe biologically isolated nucleic acids in codon sequence due to thedegeneracy of the genetic code.

According to another aspect of the invention, further isolated nucleicacid molecules that are based on the above-noted NR3B nucleic acidmolecules are provided. In this aspect, the isolated nucleic acidmolecules are selected from the group that consists of (a) a fragment ofthe nucleotide sequence set forth as SEQ ID NO: 1 between 24 and 32nucleotides in length or more, and (b) complements of (a).

The invention also provides isolated fragments of SEQ ID NO: 1 orcomplements of SEQ ID NO: 1. A fragment is one that is a ‘signature’ forthe larger nucleic acid. It, for example, is long enough to assure thatits precise sequence is not found in molecules outside of the NR3Bnucleic acid molecules defined above. Those of ordinary skill in the artmay apply no more than routine procedures to determine if a fragment iswithin the human or mouse genome. Fragments, however, exclude fragmentscompletely composed of the nucleotide sequences that are containedwithin SEQ ID NO: 1 and that are known as of the filing date of thisapplication.

Fragments can be used as probes in Southern blot, Northern blot, andGene Chip/microarray assays to identify such nucleic acid molecules, orcan be used in amplification assays such as those employing PCR As knownto those skilled in the art, large probes such as 200 nucleotides ormore are preferred for certain uses such as Southern blots, whilesmaller fragments will be preferred for uses such as in PCR and genechip/microarray assays. Fragments also can be used to produce fusionproteins for generating antibodies or determining binding of thepolypeptide fragments, or for generating immunoassay components.Likewise, fragments can be employed to produce nonfused fragments of theNR3B polypeptides that are useful, for example, in the preparation ofantibodies in immunoassays. Fragments further can be used as antisensemolecules to inhibit the expression of NR3B nucleic acids andpolypeptides, particularly for therapeutic purposes as described ingreater detail below.

As will be recognized by those skilled in the art, the size of thefragment will depend upon its conservancy in the genetic code. Thus,some regions of SEQ ID NO: 1 and its complement will require longersegments to be unique while others will require only short segments,typically between 24 and 32 nucleotides or more in length (e.g. 24, 25,26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, or more), includingevery integer up to the entire length of the disclosed sequence lessone. Many segments of the polynucleotide coding region or complementsthereof that are 18 or more nucleotides in length will be unique. Thoseskilled in the art are well versed in methods for selecting suchsequences, typically on the basis of the ability of the unique fragmentto selectively distinguish the sequence of interest from non-NR3Bnucleic acid molecules. A comparison of the sequence of the fragment tothose on known databases typically is all that is necessary, although invitro confirmatory hybridization and sequencing analysis may beperformed.

A fragment can be a functional fragment. A functional fragment of anucleic acid molecule of the invention is a fragment which retains somefunctional property of the larger nucleic acid molecule, such as codingfor a functional polypeptide, regulating cation passage in NMDAreceptors, regulating ligand binding, regulating conformation of pore,regulating transcription of operably linked nucleic acid molecules, andthe like. One of ordinary skill in the art can readily determine usingthe assays described herein and those well known in the art to determinewhether a fragment is a functional fragment of a nucleic acid moleculeusing no more than routine experimentation.

In yet another aspect of the invention, mutant NR3B nucleic acidmolecules are provided, which do not encode fully functional NR3Bpolypeptides. Rather, these mutant NR3B nucleic acid molecules of theinvention contain a sequence which is identical to SEQ ID NO: 1 with theexception that the sequence includes one or more mutations, e.g.,deletions, additions or substitutions, such that the mutant NR3B nucleicacid molecules encode mutant NR3B polypeptides, i.e., polypeptides thatdo not exhibit 100% of NR3B polypeptide functional activity. It isunderstood that some mutants will encode non-functional NR3Bpolypeptides, and other mutants will encode NR3B polypeptides withreduced or enhanced function. For example, a mutant NR3B molecule mayencode a NR3B polypeptide that has from 0 through 25% of NR3Bpolypeptide finctional activity, 26% through 50% of NR3B functionalactivity, 51% through 75% of NR3B polypeptide finctional activity, or76% through 95% of NR3B polypeptide fuctional activity, as assessed, forexample, by the electrophysiological assay for NMDA receptors and NR3Bfunction described in the Examples. It will be understood by one ofordinary skill in the art, that some mutant NR3B nucleic acids mayencode polypeptides that have over 100% of NR3B polypeptide finctionalactivity. For example, a mutant may encode a NR3B polypeptide that hasfrom 101% through 125% of NR3B functional activity, 125% through 150% ofNR3B functional activity, or 150% through 200% or more of NR3Bfunctional activity as assessed for example, by the electrophysiologicalassay described in the Examples. The level of finction of a mutant NR3Bpolypeptide can be determined and compared to that of NR3B polypeptideusing standard assays known to one of ordinary skill in the art. Suchassays include, but are not limited to the electrophysiological assaysdescribed herein. As used herein, the term “affect the functionalactivity” means to either inhibit or enhance the functional activity.

As used herein with respect to nucleic acid molecules, in general, theterm “isolated” means: (i) amplified in vitro by, for example, PCR; (ii)recombinantly produced by cloning; (iii) purified, as by cleavage andgel separation; or (iv) synthesized by, for example, chemical synthesis.An isolated nucleic acid molecule is one which is readily manipulable byrecombinant DNA techniques well known in the art. Thus, a nucleotidesequence contained in a vector in which 5′ and 3′ restriction sites areknown or for which PCR primer sequences have been disclosed isconsidered isolated but a nucleic acid sequence existing in, its nativestate in its natural host is not. An isolated nucleic acid molecule maybe substantially purified, but need not be. For example, a nucleic acidmolecule that is isolated within a cloning or expression vector is notpure in that it may comprise only a tiny percentage of the material inthe cell in which it resides. Such a nucleic acid molecule is isolated,however, as the term is used herein because it is readily manipulable bystandard techniques known to those of ordinary skill in the art. Anisolated nucleic acid molecule as used herein is not a naturallyoccurring chromosome.

As used herein, a “mutant NR3B nucleic acid molecule” refers to a NR3Bnucleic acid molecule which includes a mutation (addition, deletion, orsubstitution) such that the mutant NR3B nucleic acid molecule does notencode a fully functional NR3B polypeptide. Rather, the mutant NR3Bnucleic acid molecule encodes a mutant NR3B polypeptide, i.e., apolypeptide which does not exhibit the same functional activity as aNR3B polypeptide. Thus, a “mutant NR3B polypeptide” refers to a geneproduct of a mutant NR3B nucleic acid molecule. As used herein, the term“aberrant” includes decreased expression (including zero expression) ofthe natural NR3B molecule (nucleic acid or polypeptide) as compared toits expression in a subject who does not have a NR3B-associateddisorder, and increased expression of a mutant NR3B molecule (nucleicacid or polypeptide) as compared to its expression in a subject who doesnot have a NR3B-associated disorder.

As used herein, the term “antisense oligonucleotide” or “antisense”describes an oligonucleotide that is an oligoribonucleotide,oligodeoxyribonucleotide, modified oligoribonucleotide, or modifiedoligodeoxyribonucleotide which hybridizes under physiological conditionsto DNA comprising a particular gene or to a transcript of that gene and,thereby, inhibits the transcription of that gene and/or the translationof that mRNA. The antisense molecules are designed so as to interferewith transcription or translation of a target gene upon hybridizationwith the target gene or transcript. Those skilled in the art willrecognize that the exact length of the antisense oligonucleotide and itsdegree of complementarity with its target will depend upon the specifictarget selected, including the sequence of the target and the particularbases which comprise that sequence. It is preferred that the antisenseoligonucleotide be constructed and arranged so as to bind selectivelywith the target under physiological conditions, i.e., to hybridizesubstantially more to the target sequence than to any other sequence inthe target cell under physiological conditions. Based upon SEQ ID NO: 1or upon allelic or homologous genomic and/or cDNA sequences, one ofskill in the art can easily choose and synthesize any of a number ofappropriate antisense molecules for use in accordance with the presentinvention. In order to be sufficiently selective and potent forinhibition, such antisense oligonucleotides should comprise at least 10and, more preferably, at least 15 consecutive bases which arecomplementary to the target, although in certain cases modifiedoligonucleotides as short as 7 bases in length have been usedsuccessfully as antisense oligonucleotides (Wagner et al., NatureBiotechnology 14: 840-844, 1996).

Most preferably, the antisense oligonucleotides comprise a complementarysequence of 20-30 bases. Although oligonucleotides may be chosen whichare antisense to any region of the gene or its transcripts, in preferredembodiments the antisense oligonucleotides correspond to N-terminal or5′ upstream sites such as translation initiation, transcriptioninitiation, or promoter sites. In addition, 3′-untranslated regions maybe targeted. Targeting to mRNA splicing sites also has been used in theart but may be less preferred because alternative mRNA splicing of theNR3B transcript occurs. In addition, the antisense is targeted,preferably, to sites in which mRNA secondary structure is not expected(see, e.g., Sainio et al., Cell Mol. Neurobiol. 14(5):439-457, 1994) andat which polypeptides are not expected to bind. The present inventionalso provides for antisense oligonucleotides which are complementary togenomic DNA and/or cDNA corresponding to SEQ ID NO: 1 or SEQ ID NO: 3.Antisense to allelic or homologous cDNAs and genomic DNAs are enabledwithout undue experimentation. As mentioned above, the inventionembraces antisense oligonucleotides that selectively bind to a mutantNR3B nucleic acid molecule encoding a mutant NR3B polypeptide. This isdesirable in medical conditions wherein an aberrant NR3B expression isnot desirable.

In one set of embodiments of the aforementioned human and mousecompositions and utilities, the antisense oligonucleotides of theinvention may be composed of “natural” deoxyribonucleotides,ribonucleotides, or any combination thereof. That is, the 5′ end of onenative nucleotide and the 3′ end of another native nucleotide may becovalently linked, as in natural systems, via a phosphodiesterinternucleoside linkage. These oligonucleotides may be prepared byart-recognized methods which may be carried out manually or by anautomated synthesizer. They also may be produced recombinantly byvectors.

In preferred embodiments, however, the antisense oligonucleotides of theinvention also may include “modified” oligonucleotides. That is, theoligonucleotides may be modified in a number of ways, which do notprevent them from hybridizing to their target but which enhance theirstability or targeting or which otherwise enhance their therapeuticeffectiveness.

The term “modified oligonucleotide” as used herein describes anoligonucleotide in which (1) at least two of its nucleotides arecovalently linked via a synthetic internucleoside linkage (i.e., alinkage other than a phosphodiester linkage between the 5′ end of onenucleotide and the 3′ end of another nucleotide) and/or (2) a chemicalgroup not normally associated with nucleic acids has been covalentlyattached to the oligonucleotide. Preferred synthetic internucleosidelinkages are phosphorothioates, alkylphosphonates, phosphorodithioates,phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates,carbonates, phosphate triesters, acetamidates, carboxymethyl esters andpeptides.

The term “modified oligonucleotide” also encompasses oligonucleotideswith a covalently modified base and/or sugar. For example, modifiedoligonucleotides include oligonucleotides having backbone sugars whichare covalently attached to low molecular weight organic groups otherthan a hydroxyl group at the 3′ position and other than a phosphategroup at the 5′ position. Thus, modified oligonucleotides may include a2′-O-alkylated ribose group. In addition, modified oligonucleotides mayinclude sugars such as arabinose instead of ribose. The presentinvention, thus, contemplates pharmaceutical preparations containingmodified antisense molecules that are complementary to and hybridizablewith, under physiological conditions, nucleic acid molecules encodingNR3B polypeptides, together with pharmaceutically acceptable carriers.

Antisense oligonucleotides may be administered as part of apharmaceutical composition. Such a pharmaceutical composition mayinclude the antisense oligonucleotides in combination with any standardphysiologically and/or pharmaceutically acceptable carriers which areknown in the art. The compositions should be sterile and contain atherapeutically effective amount of the antisense oligonucleotides in aunit of weight or volume suitable for administration to a patient. Theterm “pharmaceutically acceptable” means a non-toxic material that doesnot interfere with the effectiveness of the biological activity of theactive ingredients. The term “physiologically acceptable” refers to anon-toxic material that is compatible with a biological system such as acell, cell culture, tissue, or organism. The characteristics of thecarrier will depend on the route of administration. Physiologically andpharmaceutically acceptable carriers include diluents, fillers, salts,buffers, stabilizers, solubilizers, and other materials which are wellknown in the art.

According to yet another aspect of the invention, an expression vectorcomprising any of the isolated nucleic acid molecules of the invention,preferably operably linked to a promoter is provided. In a relatedaspect, host cells transformed or transfected with such expressionvectors also are provided.

As used herein, a “vector” may be any of a number of nucleic acidmolecules into which a desired sequence may be inserted by restrictionand ligation for transport between different genetic environments or forexpression in a host cell. Vectors are typically composed of DNAalthough RNA vectors are also available. Vectors include, but are notlimited to, plasmids, phagemids and virus genomes. A cloning vector isone which is able to replicate in a host cell, and which is furthercharacterized by one or more endonuclease restriction sites at which thevector may be cut in a determinable fashion and into which a desired DNAsequence may be ligated such that the new recombinant vector retains itsability to replicate in the host cell. In the case of plasmids,replication of the desired sequence may occur many times as the plasmidincreases in copy number within the host bacterium or just a single timeper host before the host reproduces by mitosis. In the case of phage,replication may occur actively during a lytic phase or passively duringa lysogenic phase.

An expression vector is one into which a desired DNA sequence may beinserted by restriction and ligation such that it is operably joined toregulatory sequences and may be expressed as an RNA transcript. Vectorsmay further contain one or more marker sequences suitable for use in theidentification of cells which have or have not been transformed ortransfected with the vector. Markers include, for example, genesencoding polypeptides which increase or decrease either resistance orsensitivity to antibiotics or other compounds, genes which encodeenzymes whose activities are detectable by standard assays known in theart (e.g., β-galactosidase, alkaline phosphatase, or luciferase), andgenes which visibly affect the phenotype of transformed or transfectedcells, hosts, colonies or plaques (e.g., green fluorescent protein).Preferred vectors are those capable of autonomous replication andexpression of the structural gene products present in the DNA segmentsto which they are operably joined.

As used herein, a coding sequence and regulatory sequences are said tobe “operably” joined when they are covalently linked in such a way as toplace the expression or transcription of the coding sequence under theinfluence or control of the regulatory sequences. If it is desired thatthe coding sequences be translated into a functional polypeptide, twoDNA sequences are said to be operably joined if induction of a promoterin the 5′ regulatory sequences results in the transcription of thecoding sequence and if the nature of the linkage between the two DNAsequences does not (1) result in the introduction of a frame-shiftmutation, (2) interfere with the ability of the promoter region todirect the transcription of the coding sequences, or (3) interfere withthe ability of the corresponding RNA transcript to be translated into apolypeptide. Thus, a promoter region would be operably joined to acoding sequence if the promoter region were capable of effectingtranscription of that DNA sequence such that the resulting transcriptmight be translated into the desired protein or polypeptide.

The precise nature of the regulatory sequences needed for geneexpression may vary between species or cell types, but shall in generalinclude, as necessary, 5′ non-transcribed and 5′ non-translatedsequences involved with the initiation of transcription and translationrespectively, such as a TATA box, capping sequence, CAAT sequence, andthe like. Especially, such 5′ non-transcribed regulatory sequences willinclude a promoter region which includes a promoter sequence fortranscriptional control of the operably joined gene. Regulatorysequences may also include enhancer sequences or upstream activatorsequences as desired. The vectors of the invention may optionallyinclude S′ leader or signal sequences. The choice and design of anappropriate vector is within the ability and discretion of one ofordinary skill in the art.

It will also be recognized that the invention embraces the use of theNR3B cDNA sequences or mutant NR3B cDNA sequences in expression vectors,as well as to transfect host cells and cell lines, be these prokaryotic(e.g., E. coli), or eukaryotic (e.g., CHO cells, COS cells, HEK293cells, Xenopus oocytes, yeast expression systems and recombinantbaculovirus expression in insect cells). Especially useful are mammaliancells such as human, mouse, hamster, pig, goat, primate, etc. They maybe of a wide variety of tissue types, including neuronal cells,fibroblasts, oocytes, monocytes, lymphocytes, and they may be primarycells or cell lines. Specific examples include keratinocytes, neuronalcells, and embryonic stem cells. The expression vectors require that thepertinent sequence, i.e., those nucleic acids described herein, beoperably linked to a promoter.

In some embodiments, NR3B subunit-encoding DNA is ligated into a vector,and introduced into suitable host cells to produce transformed celllines that express a specific NMDA receptor subtype, or specificcombinations of subunits, including NR3B. The resulting cell lines canthen be produced in quantity for reproducible quantitative analysis ofthe effects of known or potential drugs on receptor function. In otherembodiments, mRNA may be produced by in vitro transcription of DNAencoding each subunit. This mRNA, either from a single subunit clone orfrom a combination of clones, can then be injected into Xenopus oocytesusing standard procedures known to those of ordinary skill in the art,where the mRNA directs the synthesis of the receptor subunits, whichthen form functional receptors. Alternatively, the subunit-encoding DNAcan be directly injected into oocytes for expression of functionalreceptors. The transfected mammalian cells or injected oocytes may thenbe used in the methods of drug screening provided herein.

Eukaryotic cells in which DNA or RNA may be introduced include any cellsthat are transfectable by such DNA or RNA or into which such DNA or RNAmay be injected. Preferred cells are those that can be transiently orstably transfected and also express the DNA and RNA. Presently mostpreferred cells are those that can form recombinant or heterologous NMDAreceptors comprising one or more subunits encoded by the heterologousDNA. Such cells may be identified empirically or selected from amongthose known to be readily transfected or injected.

Exemplary cells for introducing DNA include cells of mammalian origin(e.g., COS cells, mouse L cells, Chinese hamster ovary (CHO) cells,human embryonic kidney (HEK) cells (particularly HBK293 cells that canbe frozen in liquid nitrogen and then thawed and regrown; for example,those described in U.S. Pat. No. 5,024,939 to Gorman (see, also,Stillman et al. (1985) Mol. Cell. Biol. 5:2051-2060)), African greenmonkey cells (and other such cells known to those of skill in the art),amphibian cells (e.g., Xenopus laevis oocytes), yeast cells (e.g.,Saccharomyces cerevisiae, Pichia pastoris), and the like. Exemplarycells for expressing injected RNA transcripts include Xenopus laevisoocytes. Cells for transfection of DNA are known to those of skill inthe art or may be empirically identified, and include HEK293 (which areavailable from ATCC under accession #CRL 1573); Ltk⁻¹ cells (which areavailable from ATCC under accession #CCL1.3); COS-7 cells (which areavailable from ATCC under accession #CRL 1651); and DG44 cells (dhfr CHOcells; see, e.g., Urlaub et al. (1986) Cell. Molec. Genet. 12:555).

DNA may be stably incorporated into cells or may be transientlyexpressed using methods known in the art. Stably transfected mammaliancells may be prepared by transfecting cells with an expression vectorhaving a selectable marker gene, and growing the transfected cells underconditions selective for cells expressing the marker gene. To preparetransient transfectants, mammalian cells are transfected with a reportergene (such as the E. coli β-galactosidase gene) to monitor transfectionefficiency. Selectable marker genes usually are not included in thetransient transfections because the transfectants are typically notgrown under selective conditions, and are usually analyzed within a fewdays after transfection.

To produce such stably or transiently transfected cells, the cellsshould be transfected with a sufficient concentration ofsubunit-encoding nucleic acids to form NMDA receptors that contain thesubunits encoded by heterologous DNA. The precise amounts and ratios ofDNA encoding the subunits may be empirically determined and optimizedfor a particular combination of subunits, cells and assay conditions.Recombinant cells that express NMDA receptors containing subunitsencoded only by the heterologous DNA or RNA are especially preferred.

Heterologous DNA may be maintained in the cell as an episomal element ormay be integrated into chromosomal DNA of the cell. The resultingrecombinant cells may then be cultured or subcultured (or passaged, inthe case of mammalian cells) from such a culture or a subculturethereof. Methods for transfection, injection and culturing recombinantcells are known to the skilled artisan.

As used herein, the terms “heterologous” or foreign” DNA and RNA areused interchangeably and refer to DNA or RNA that does not occurnaturally as part of the genome of the cell in which it is present or toDNA or RNA that is found in a location or locations in the genome thatdiffer from that in which it occurs in nature. Typically, heterologousor foreign DNA and RNA refers to DNA or RNA that is not endogenous tothe host cell and has been artificially introduced into the cell.Examples of heterologous DNA include DNA that encodes a NMDA receptorsubunit, DNA that encodes RNA or polypeptides that mediate or alterexpression of endogenous DNA by affecting transcription, translation, orother regulatable biochemical processes, and the like. The cell thatexpresses heterologous DNA may contain DNA encoding the same ordifferent expression products. Heterologous DNA need not be expressedand may be integrated into the host cell genome or maintainedepisomally.

Recombinant receptors on eukaryotic cell surfaces may contain one ormore subunits encoded by the DNA or mRNA encoding NMDA receptorsubunits, or may contain a mixture of subunits encoded by the host celland subunits encoded by heterologous DNA or mRNA. Recombinant receptorsmay be homomeric or may be a heteromeric combination of multiplesubunits. Mixtures of DNA or mRNA encoding receptors subunits fromvarious species, such as mice and humans, may also be introduced intothe cells. Thus, a cell can be prepared that expresses recombinantreceptors containing only NR3B subunits, or a combination of any one ormore NR1, NR2A-NR2D, or NR3A and any one or more NR3B subunits providedherein. For example, NR3B subunits of the present invention can beco-expressed with NR1 and/or NR2A receptor subunits. Specific examplesof heteromeric combinations of recombinant human NR3B subunits that havebeen expressed in HEK293 cells include NR3B+NR1 and NR3B+NR2A (seeExamples).

In general, the conditional expression vectors used in such systems usea variety of promoters which confer the desired gene expression pattern(e.g., temporal or spatial). Conditional promoters also can be operablylinked to NR3B nucleic acid molecules to increase or decrease expressionof a NR3B molecule in a regulated or conditional maimer. Trans-actingnegative or positive regulators of NR3B activity or expression also canbe operably linked to a conditional promoter as described above. Suchtrans-acting regulators include antisense NR3B nucleic acid molecules,nucleic acid molecules which encode dominant negative NR3B molecules,ribozyme molecules specific for NR3B nucleic acid molecules, and thelike. (see Sze, S. C., et al., Neurochem Int. October2001;39(4):319-27). The transgenic non-human animals are useful interalia, for testing biochemical or physiological effects of diagnostics ortherapeutics for conditions characterized by altered NR3B moleculeexpression or function. Other uses will be apparent to one of ordinaryskill in the art. Thus, the invention also permits the construction ofNR3B gene “knock-outs” in cells and in animals, providing materials forstudying certain aspects of NMDA receptor disorders and/orNR3B-associated motor neuron diseases.

The invention also permits the construction of NR3B polypeptide gene“knock-outs” or “knock-ins” in cells and in animals, providing materialsfor studying certain aspects of NR3B-associated motor neuron diseasesand immune system responses to NR3B-associated motor neuron diseases byregulating the expression of NR3B polypeptides. For example, a knock-inmouse may be constructed and examined for clinical parallels between themodel and a NR3B-associated motor neuron disease affected mouse withupregulated expression of a normal or mutated NR3B polypeptide. Such acellular or animal model may be useful for assessing treatmentstrategies for NR3B -associated motor neuron diseases. An example of a“knock-in” mouse, although not intended to be limiting, involvesintroducing a NR3B molecule into the cell line and introducing that cellline into a mouse. A “knock-in” model provides a model with which toevaluate the effects of candidate pharmaceutical compounds (e.g.inhibitory effects) on a living animal that expresses a NR3B molecule.

Alternative types of animal models for NR3B-associated motor neurondiseases may be developed based on the invention and may provide a modelin which to test treatments, and assess the etiology of NR3B-associatedmotor neuron diseases.

According to another aspect of the invention, a transgenic non-humananimal comprising an expression vector of the invention is provided,including a transgenic non-human animal which has reduced expression ofa NR3B nucleic acid molecule or elevated expression of a NR3B or mutantNR3B nucleic acid molecule.

Thus the transgenic animal include “knock-out” animals having ahomozygous or heterozygous gene disruption by homologous recombination,animals having episomal or chromosomally incorporated expressionvectors, etc. Knock-out animals can be prepared by homologousrecombination using embryonic stem cells as is well known in the art.The recombination can be facilitated by the cre/lox system or otherrecombinase systems known to one of ordinary skill in the art. Incertain embodiments, the recombinase system itself is expressedconditionally, for example, in certain tissues or cell types, at certainembryonic or post-embryonic developmental stages, inducibly by theaddition of a compound which increases or decreases expression, and thelike.

The invention also embraces so-called expression kits, which allow theartisan to prepare a desired expression vector or vectors. Suchexpression kits include at least separate portions of each of thepreviously discussed coding sequences. Other components may be added, asdesired, as long as the previously mentioned sequences, which arerequired, are included.

According to another aspect of the invention, an isolated NR3Bpolypeptide encoded by any of the foregoing isolated nucleic acidmolecules of the invention is provided. As used herein, a NR3Bpolypeptide refers to a polypeptide which is encoded by a nucleic acidhaving SEQ ID NO: 1, a functional fragment thereof, or a functionalequivalent thereof (e.g., a nucleic acid sequence encoding the samepolypeptide as encoded by SEQ ID NO: 1), provided that the functionalfragment or equivalent encodes a NR3B polypeptide which exhibits a NR3Bfunctional activity. As used herein, a NR3B functional activity refersto the ability of a NR3B polypeptide to modulate one or more of thefollowing parameters: NMDA receptor activity, cation passage in NMDAreceptor channel, and/or NMDA receptor ligand binding. An exemplary NR3Bfunctional activity is modulation of NMDA receptor activity and ion flowfollowing ligand binding to a NMDA receptor. NR3B functional activitycan be determined by measuring electrophysiological changes in cationpassage at the NMDA receptor using, for example, the assays described inthe Examples.

Preferably, the isolated polypeptide comprises the amino acid sequenceset forth as: SEQ ID NO: 2 or fragments of SEQ ID NO: 2. Suchpolypeptides are useful, for example, alone or as fusion polypeptides togenerate antibodies for NR3B polypeptides. The polypeptides of theinvention are also useful in animal models of motor neuron disease, forexample, an NR3B polypeptide of the invention may be expressed as areceptor subunit in a NMDA receptor and its expression and/or functionmay be monitored using methods described herein to determine thefunction of the NR3B polypeptide NMDA receptor subunit in motor neurondisease. In addition, expression and/or function of the NR3B polypeptideof the invention can be modulated using methods described herein and theeffect on the NMDA receptor activity can be evaluated as a method ofdetermining the disease process in motor neuron disease such as ALS. Inaddition, the multimeric NMDA receptors containing NR3B and othersubunits can be generated and expressed in cells, for example cells inculture, using methods described herein. These cells may be useful forexamining functional activity variations of different subunitcombinations in NMDA receptors, and may also be useful in screeningassays for evaluating the effect of candidate pharmacological compoundson function of a NR3B subunit of a glutamate receptor. The animal modelsincluding NR3B polypeptide NMDA receptor subunits of the invention arealso useful for such screening assays.

The invention also provides functional polypeptide fragments, andfunctional combinations thereof, encoded by the DNAs of the invention.Such functional polypeptide fragments can be produced by those skilledin the art, without undue experimentation, by eliminating some or all ofthe amino acids in the sequence not essential for the polypeptide tofunction as a glutamate receptor subunit. A determination of the aminoacids that are essential for glutamate receptor function is made, forexample, by systematic digestion of the DNAs encoding the polypeptidesand/or by the introduction of deletions into the DNAs. The modified(e.g., deleted or digested) DNAs are expressed, for example, bytranscribing the DNA and then introducing the resulting mRNA intoXenopus oocytes, where translation of the mRNAs will occur. Functionalanalysis of the polypeptides thus expressed in the oocytes isaccomplished by exposing the oocytes to ligands known to bind to andfunctionally activate glutamate receptors, and then monitoring theoocytes to see if the expressed fragments form ion channel(s). If ionchannel(s) are detected, the fragments are functional as glutamatereceptor subunits. (see U.S. Pat. No. 6,111,091)

Polypeptides can be isolated from biological samples including tissue orcell homogenates, and can also be expressed recombinantly in a varietyof prokaryotic and eukaryotic expression systems by constructing anexpression vector appropriate to the expression system, introducing theexpression vector into the expression system, and isolating therecombinantly expressed polypeptide. Short polypeptides, includingantigenic peptides (such as those presented by MHC molecules on thesurface of a cell for immune recognition) also can be synthesizedchemically using well-established methods of peptide synthesis.

Thus, as used herein with respect to polypeptides, “isolated” meansseparated from its native environment and present in sufficient quantityto permit its identification or use. Isolated, when referring to aprotein or polypeptide, means, for example: (i) selectively produced byexpression of a recombinant nucleic acid or (ii) purified as bychromatography or electrophoresis. Isolated proteins or polypeptidesmay, but need not be, substantially pure. The term “substantially pure”means that the proteins or polypeptides are essentially free of othersubstances with which they may be found in nature or in vivo systems toan extent practical and appropriate for their intended use.Substantially pure polypeptides may be produced by techniques well knownin the art. Because an isolated polypeptide may be admixed with apharmaceutically acceptable carrier in a pharmaceutical preparation, thepolypeptide may comprise only a small percentage by weight of thepreparation. The polypeptide is nonetheless isolated in that it has beenseparated from the substances with which it may be associated in livingsystems, e.g. isolated from other polypeptides.

A fragment of a NR3B polypeptide, for example, generally has thefeatures and characteristics of fragments including unique fragments asdiscussed above in connection with nucleic acid molecules. As will berecognized by those skilled in the art, the size of a fragment which isunique will depend upon factors such as whether the fragment constitutesa portion of a conserved protein domain. Thus, some regions of NR3Bpolypeptide, for example a conserved binding domain, may require longersegments to be unique. Others will require only short segments,typically between 5 and 14 amino acids (e.g., 5, 6, 7, 8, 9, 10, 11, 12,13, and 14 amino acids long) as used to generate NR3B-specificantibodies.

Fragments of a polypeptide preferably are those fragments which retain adistinct functional capability of the polypeptide. Functionalcapabilities which can be retained in a fragment of a polypeptideinclude interaction with antibodies, interaction with other polypeptidesor fragments thereof, selective binding of nucleic acid molecules, andenzymatic activity. Functional activity of the NMDA receptor as a wholecan also be assessed with respect to the identity of fragments of NR3Bthat allow the NMDA receptor to maintain normal function. For example,fragments can be tested in a cell or animal model to determine the sizeand sequence of fragments that when expressed as a subunit of an NMDAreceptor, allow normal or aberrant function of that receptor. Oneimportant activity is the ability to act as a signature for identifyingthe polypeptide. Another is the ability to provoke an immune response toa mutant NR3B molecule but not provoke an immune response to normallevels of a nonmutated NR3B molecule. For example, identification of afragment of a mutant NR3B subunit that is antigenic, in contrast to anon-antigenic normal NR3B subunit.

Those skilled in the art are well versed in methods for selecting uniqueamino acid sequences, typically on the basis of the ability of thefragment to selectively distinguish the sequence of interest fromnon-family members. A comparison of the sequence of the fragment tothose on known databases typically is all that is necessary.

The invention embraces variants of the NR3B polypeptides describedherein. As used herein, a “variant” of a NR3B polypeptide is apolypeptide which contains one or more modifications to the primaryamino acid sequence of a NR3B polypeptide. Modifications which create aNR3B polypeptide variant can be made to a NR3B polypeptide 1) increase,reduce, or eliminate an activity of the NR3B polypeptide; 2) to enhancea property of the NR3B polypeptide, such as polypeptide stability in anexpression system or the stability of protein-protein binding; or 3) toprovide a novel activity or property to a NR3B polypeptide, such asaddition of an antigenic epitope or addition of a detectable moiety.

Modifications to a NR3B polypeptide are typically made to the nucleicacid molecule which encodes the polypeptide, and can include deletions,point mutations, truncations, amino acid substitutions, and additions ofamino acids or non-amino acid moieties. Alternatively, modifications canbe made directly to the polypeptide, such as by cleavage, addition of alinker molecule, addition of a detectable moiety, such as biotin,addition of a fatty acid, and the like. Modifications also embracefusion proteins comprising all or part of the NR3B amino acid sequences.One of skill in the art will be familiar with methods for predicting theeffect on polypeptide conformation of a change in polypeptide sequence,and can thus “design” a variant NR3B polypeptide according to knownmethods. One example of such a method is described by Dahiyat and Mayoin Science 278:82-87, 1997, whereby polypeptides can be designed denovo. The method can be applied to a known polypeptide to vary only aportion of the polypeptide sequence. By applying the computationalmethods of Dahiyat and Mayo, specific variants of a NR3B polypeptide canbe proposed and tested to determine whether the variant retains adesired conformation.

In general, variants include NR3B polypeptides which are modifiedspecifically to alter a feature of the polypeptide unrelated to itsdesired physiological activity. For example, cysteine residues can besubstituted or deleted to prevent unwanted disulfide linkages.Similarly, certain amino acids can be changed to enhance expression of aNR3B polypeptide by eliminating proteolysis by proteases in anexpression system (e.g., dibasic amino acid residues in yeast expressionsystems in which KEX2 protease activity is present).

Mutations of a nucleic acid molecule which encode a NR3B polypeptidepreferably preserve the amino acid reading frame of the coding sequence,and preferably do not create regions in the nucleic acid which arelikely to hybridize to form secondary structures, such a hairpins orloops, which can be deleterious to expression of the variantpolypeptide.

Mutations can be made by selecting an amino acid substitution, or byrandom mutagenesis of a selected site in a nucleic acid which encodesthe polypeptide. Variant polypeptides are then expressed and tested forone or more activities to determine which mutation provides a variantpolypeptide with the desired properties. Further mutations can be madeto variants (or to non-variant NR3B polypeptides) which are silent as tothe amino acid sequence of the polypeptide, but which provide preferredcodons for translation in a particular host. The preferred codons fortranslation of a nucleic acid in, e.g., E. coli, are well known to thoseof ordinary skill in the art. Still other mutations can be made to thenoncoding sequences of a NR3B gene or cDNA clone to enhance expressionof the polypeptide. The activity of variants of NR3B polypeptides can betested by cloning the gene encoding the variant NR3B polypeptide into abacterial, amphibian, or mammalian expression vector, introducing thevector into an appropriate host cell, expressing the variant NR3Bpolypeptide, and testing for a functional capability of the NR3Bpolypeptide as disclosed herein. Preparation of other variantpolypeptides may favor testing of other activities, as will be known toone of ordinary skill in the art.

The skilled artisan will also realize that conservative amino acidsubstitutions may be made in NR3B polypeptides to provide functionalvariants of the foregoing polypeptides, i.e., the variants which thefunctional capabilities of the NR3B polypeptides. As used herein, a“conservative amino acid substitution” refers to an amino acidsubstitution which does not alter the relative charge or sizecharacteristics of the polypeptide in which the amino acid substitutionis made. Conservative substitutions of amino acids include substitutionsmade amongst amino acids within the following groups: (a) M, I, L, V;(b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

For example, upon determining that a polypeptide derived from a NR3Bpolypeptide possesses modulator activity such as suppressing/reducingNMDA receptor function and/or cation passage, one can make conservativeamino acid substitutions to the amino acid sequence of the polypeptide.The substituted polypeptides can then be tested for one or more of theabove-noted functions, in vivo or in vitro. These variants can be testedfor improved stability and are useful, inter alia, in pharmaceuticalcompositions.

Functional variants of NR3B polypeptides, i.e., variants of polypeptideswhich retain the function of the NR3B polypeptides, can be preparedaccording to methods for altering polypeptide sequence known to one ofordinary skill in the art such as are found in references which compilesuch methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook,et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Exemplaryfunctional variants of the NR3B polypeptides include conservative aminoacid substitutions of the polypeptides encoded by SEQ ID NO: 2 and SEQID NO: 4. Conservative amino-acid substitutions in the amino acidsequence of NR3B polypeptides to produce functional variants of NR3Bpolypeptides typically are made by alteration of the nucleic acidmolecule encoding a NR3B polypeptide (e.g., SEQ ID NO:1). Suchsubstitutions can be made by a variety of methods known to one ofordinary skill in the art. For example, amino acid substitutions may bemade by PCR-directed mutation, site-directed mutagenesis according tothe method of Kunkel (Kunkel, Proc. Nat. Acad. Sci. U.S.A. 82: 488-492,1985), or by chemical synthesis of a gene encoding a NR3B polypeptide.Where amino acid substitutions are made to a small unique fragment of aNR3B polypeptide, the substitutions can be made by directly synthesizingthe polypeptide. The activity of functional variants or fragments ofNR3B polypeptide can be tested by cloning the gene or transcript thatencodes the altered NR3B polypeptide into a bacterial, mammalian, orinsect cell expression vector, introducing the vector into anappropriate host cell, expressing the altered NR3B polypeptide, (or byexpressing the altered NR3B in oocytes as described herein) and testingfor a functional capability of the NR3B polypeptides as disclosedherein.

The invention as described herein has a number of uses, some of whichare described elsewhere herein. First, the invention permits isolationof the NR3B polypeptide molecules. A variety of methodologies well knownto the skilled practitioner can be utilized to obtain isolated NR3Bmolecules. The polypeptide may be purified from cells which naturallyproduce the polypeptide by chromatographic means or immunologicalrecognition. Alternatively, an expression vector may be introduced intocells to cause production of the polypeptide. In another method, mRNAtranscripts may be microinjected or otherwise introduced into cells tocause production of the encoded polypeptide, as described herein.Translation of mRNA in cell-free extracts such as the reticulocytelysate system also may be used to produce polypeptide. Those skilled inthe art also can readily follow known methods for isolating NR3Bpolypeptides. These include, but are not limited to, HPLC,size-exclusion chromatography, ion-exchange chromatography, andimmune-affinity chromatography. In addition, those skilled in the artwill also be able to utilize recombinant NR3B to determine the structure(e.g. quaternary, tertiary) of the NR3B polypeptide or its variants.

The isolation and identification of NR3B nucleic acid molecules alsomakes it possible for the artisan to diagnose a disorder characterizedby aberrant expression or function of a NR3B nucleic acid molecule orpolypeptide. These methods involve determining the aberrant expressionof one or more NR3B nucleic acid molecules and/or encoded NR3Bpolypeptides. In the former situations, such determinations can becarried out via any standard nucleic acid determination assay, includingthe PCR, or assaying with labeled hybridization probes. In the lattersituations, such determinations can be carried out by screening patientantisera for recognition of the polypeptide or by assaying biologicalsamples with binding partners (e.g., antibodies) for NR3B polypeptides.

The invention also provides, in certain embodiments, “dominant negative”polypeptides derived from NR3B polypeptides. A dominant negativepolypeptide is an inactive variant of a polypeptide, which, byinteracting with the cellular machinery, displaces an active polypeptidefrom its interaction with the cellular machinery or competes with theactive polypeptide, thereby reducing the effect of the activepolypeptide. Dominant negative polypeptides are useful, or example, forpreparing transgenic non-human animals to further characterize thefunctions of the NR3B molecules disclosed herein. For example, adominant negative receptor which binds a ligand but does not transmit asignal in response to binding of the ligand can reduce the biologicaleffect of expression of the ligand. Similarly, a dominant negativetranscription factor which binds to a promoter site in the controlregion of a gene but does not increase gene transcription can reduce theeffect of a normal transcription factor by occupying promoter bindingsites without increasing transcription. Another example of a dominantnegative NR3B is a mutant form of this polypeptide lacking aligand-binding domain.

The end result of the expression of a dominant negative polypeptide in acell is an altered function of active polypeptides. One of ordinaryskill in the art can assess the potential for a dominant negativevariant of a polypeptide, and using standard mutagenesis techniques tocreate one or more dominant negative variant polypeptides. For example,one of ordinary skill in the art can modify the sequence of NR3Bpolypeptides by site-specific mutagenesis, scanning mutagenesis, partialgene deletion or truncation, and the like. See, e.g., U.S. Pat. No.5,580,723 and Sambrook et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor Laboratory Press, 1989. The skilledartisan then can test the population of mutagenized polypeptides fordiminution in a selected and/or for retention of such an activity. Othersimilar methods for creating and testing dominant negative variants of apolypeptide will be apparent to one of ordinary skill in the art.

In yet a further aspect of the invention, binding polypeptides thatselectively bind to a NR3B molecule are provided. According to thisaspect, the binding polypeptides bind to an isolated nucleic acid orpolypeptide of the invention, including binding to unique fragmentsthereof. Preferably, the binding polypeptides bind to a NR3B polypeptideor a fragment thereof. In certain particularly preferred embodiments,the binding polypeptide binds to a mutant NR3B polypeptide but does notbind to a non-mutant NR3B polypeptide, i.e., the binding polypeptidesare selective for binding to the mutant NR3B polypeptide and can be usedin various assays to detect the presence of the mutant NR3B polypeptidewithout detecting non-mutant NR3B polypeptide. Such mutant N3Bpolypeptide binding polypeptides also can be used to selectively bind toa mutant NR3B molecule in a cell (in vivo or ex vivo) for imaging andtherapeutic applications in which, for example, the binding polypeptideis tagged with a detectable label and/or a toxin for targeted deliveryto the mutant NR3B molecule. In other preferred embodiments, the bindingpolypeptide binds to a NR3B polypeptide but does not bind to a mutantNR3B polypeptide, i.e., the binding polypeptides are selective forbinding to the NR3B polypeptide and can be used in various assays todetect the presence of the NR3B polypeptide without detecting mutantNR3B polypeptide.

In preferred embodiments, the binding polypeptide is an antibody orantibody fragment, such as an Fab or F(ab)₂ fragment of an antibody.Typically, the fragment includes a CDR3 region that is selective for theNR3B polypeptide. Any of the various types of antibodies can be used forthis purpose, including monoclonal antibodies, humanized antibodies, andchimeric antibodies.

Thus, the invention provides agents which bind to NR3B polypeptidesencoded by NR3B nucleic acid molecules, respectively, and in certainembodiments preferably to unique fragments of the NR3B polypeptides.Such binding partners can be used in screening assays to detect thepresence or absence of a NR3B polypeptide and in purification protocolsto isolate such NR3B polypeptides. Likewise, such binding partners canbe used to selectively target drugs, toxins or other molecules to cellswhich express mutant NR3B polypeptides. In this manner, cells present intissues that express mutant NR3B polypeptides can be treated withcytotoxic compounds. Such agents also can be used to inhibit the nativeactivity of the NR3B polypeptides, for example, by binding to suchpolypeptides, to further characterize the functions of these molecules.

In addition, antibodies generated against the above-described NR3B andmutant NR3B polypeptides are provided. Such antibodies can be employedfor studying receptor tissue localization, subunit composition,structure of functional domains, as well as in diagnostic applications,therapeutic applications, and the like

Factors to consider in selecting portions of the NR3B subunit for use asan immunogen (as either a synthetic peptide or a recombinantly producedbacterial fusion protein) include antigenicity, accessibility (i.e.,extracellular and cytoplasmic domains), uniqueness to the particularsubunit, etc.

The availability of subunit-specific antibodies makes possible theapplication of the technique of immunohistochemistry to monitor thedistribution and expression density of NR3B (e.g., in normal versusdiseased brain tissue). Such antibodies could also be employed fordiagnostic and therapeutic applications. For example, invention providesmethods for modulating the ion channel activity of receptor(s) of theinvention by contacting NR3B subunit(s) with an effective amount of theabove-described antibodies.

The invention, therefore, provides antibodies or fragments of antibodieshaving the ability to selectively bind to NR3B polypeptides, andpreferably to unique fragments thereof. Antibodies include polyclonal,monoclonal, and chimeric antibodies, prepared, e.g., according toconventional methodology.

The antibodies of the present invention are prepared by any of a varietyof methods, including administering polypeptide, fragments ofpolypeptide, cells expressing the polypeptide or fragments thereof andthe like to an animal to induce polyclonal antibodies. Monoclonalantibodies are produced according to techniques well known in the art.As detailed herein, such antibodies may be used for example to identifytissues expressing polypeptide or to purify polypeptide.

Significantly, as is well known in the art, only a small portion of anantibody molecule, the paratope, is involved in the binding of theantibody to its epitope (see, in general, Clark, W. R. (1986) TheExperimental Foundations of Modern Immunonology Wiley & Sons, Inc., NewYork; Roitt, I. (1991) Essential Immunology 7th Ed., BlackwellScientific Publications, Oxford). The pFc′ and Fc regions, for example,are effectors of the complement cascade but are not involved in antigenbinding. An antibody from which the pFc′ region has been enzymaticallycleaved, or which has been produced without the pFc′ region, designatedan F(ab′)₂ fragment, retains both of the antigen binding sites of anintact antibody. Similarly, an antibody from which the Fc region hasbeen enzymatically cleaved, or which has been produced without the Fcregion, designated an Fab fragment, retains one of the antigen bindingsites of an intact antibody molecule. Proceeding further, Fab fragmentsconsist of a covalently bound antibody light chain and a portion of theantibody heavy chain denoted Fd. The Fd fragments are the majordeterminant of antibody specificity (a single Fd fragment may beassociated with up to ten different light chains without alteringantibody specificity) and Fd fragments retain epitope-binding ability inisolation.

Within the antigen-binding portion of an antibody, as is well-known inthe art, there are complementarity determining regions (CDRs), whichdirectly interact with the epitope of the antigen, and framework regions(FRs), which maintain the tertiary structure of the paratope (see, ingeneral, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragmentand the light chain of IgG immunoglobulins, there are four frameworkregions (FR1 through FR4) separated respectively by threecomplementarity determining regions (CDR1 through CDR3). The CDRs, andin particular the CDR3 regions, and more particularly the heavy chainCDR3, are largely responsible for antibody specificity.

It is now well established in the art that the non-CDR regions of amammalian antibody may be replaced with similar regions of nonspecificor heterospecific antibodies while retaining the epitopic specificity ofthe original antibody. This is most clearly manifested in thedevelopment and use of “humanized” antibodies in which non-human CDRsare covalently joined to human FR and/or Fc/pFc′ regions to produce afunctional antibody. (see, e.g., U.S. Pat. Nos. 4,816,567, 5,225,539,5,585,089, 5,693,762, and 5,859,205). Thus, for example, PCTInternational Publication Number WO 92/04381 teaches the production anduse of humanized murine RSV antibodies in which at least a portion ofthe murine FR regions have been replaced by FR regions of human origin.Such antibodies, including fragments of intact antibodies withantigen-binding ability, are often referred to as “chimeric” antibodies.

Fully human monoclonal antibodies also can be prepared by immunizingmice transgenic for large portions of human immunoglobulin heavy andlight chain loci. -Following immunization of-these mice (e.g., XenoMouse(Abgenix), HuMAb mice (Medarex/GenPharm)), monoclonal antibodies can beprepared according to standard hybridoma technology. These monoclonalantibodies will have human immunoglobulin amino acid sequences andtherefore will not provoke human anti-mouse antibody (HAMA) responseswhen administered to humans.

Thus, as will be apparent to one of ordinary skill in the art, thepresent invention also provides for F(ab′)₂, Fab, Fv and Fd fragments;chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2and/or light chain CDR3 regions have been replaced by homologous humanor non-human sequences; chimeric F(ab′)₂ fragment antibodies in whichthe FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have beenreplaced by homologous human or non-human sequences; chimeric Fabfragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or lightchain CDR3 regions have been replaced by homologous human or non-humansequences; and chimeric Fd fragment antibodies in which the FR and/orCDR1 and/or CDR2 regions have been replaced by homologous human ornon-human sequences. The present invention also includes so-calledsingle chain antibodies. An anti-peptide approach is another methodologythat can be used to generate new antibodies.

Thus, the invention involves polypeptides of numerous size and type thatbind specifically to NR3B polypeptides. These polypeptides may bederived also from sources other than antibody technology. For example,such polypeptide binding agents can be provided by degenerate peptidelibraries which can be readily prepared in solution, in immobilized formor as phage display libraries. Combinatorial libraries also can besynthesized of peptides containing one or more amino acids. Librariesfurther can be synthesized of peptides and non-peptide syntheticmoieties.

Phage display can be particularly effective in identifying bindingpeptides useful according to the invention. Briefly, one prepares aphage library (using e.g. m13, fd, or lambda phage), displaying insertsfrom 4 to about 80 amino acid residues using conventional procedures.The inserts may represent a completely degenerate or biased array. Onethen can select phage-bearing inserts which bind to a NR3B polypeptide.This process can be repeated through several cycles of reselection ofphage that bind to a NR3B polypeptide. Repeated rounds lead toenrichment of phage bearing particular sequences. DNA sequence analysiscan be conducted to identify the sequences of the expressedpolypeptides. The minimal linear portion of the sequence that binds tothe NR3B polypeptide can be determined. One can repeat the procedureusing a biased library containing inserts containing part or all of theminimal linear portion plus one or more additional degenerate residuesupstream or downstream thereof. Thus, the NR3B polypeptides of theinvention can be used to screen peptide libraries, including phagedisplay libraries, to identify and select peptide binding partners ofthe NR3B polypeptides of the invention. Such molecules can be used, asdescribed, for screening assays, for diagnostic assays, for purificationprotocols or for targeting drugs, toxins and/or labeling agents (e.g.radioisotopes, fluorescent molecules, etc). to cells which expressmutant NR3B genes such as neuronal cells which have aberrant NR3Bexpression.

As detailed herein, the foregoing antibodies and other binding moleculesmay be used for example to identify tissues expressing NR3B polypeptideor to purify NR3B polypeptide. Antibodies also may be coupled tospecific diagnostic labeling agents for imaging NR3B expression in cellsand tissues, and coupled to therapeutically useful agents according tostandard coupling procedures. Diagnostic agents include, but are notlimited to, barium sulfate, iocetamic acid, iopanoic acid, ipodatecalcium, diatrizoate sodium, diatrizoate meglumine, metrizamide,tyropanoate sodium and radiodiagnostics including positron emitters suchas fluorine-18 and carbon-11, gamma emitters such as iodine-123,technitium-99m, iodine-131 and indium-111, nuclides for nuclear magneticresonance such as fluorine and gadolinium. Other diagnostic agentsuseful in the invention will be apparent to one of ordinary skill in theart.

In some circumstances, it may be preferred to conjugate molecules to acompound which facilitates transport of the molecule across theblood-brain barrier (BBB) for transport into the central nervous system.Such molecules for transport may include, but are not limited to: NR3B,NR3B-binding polypeptide (for example an anti-NR3B antibody), NMDAreceptor agonist, NMDA receptor antagonist, and the like. As usedherein, a compound which facilitates transport across the BBB is onewhich, when conjugated to the molecule to be transported, facilitatesthe amount of that molecule delivered to the brain as compared withnon-conjugated molecule. The compound can induce transport across theBBB by any mechanism, including receptor-mediated transport, anddiffusion. The molecule to be transported can be conjugated to suchcompounds by well-known methods, including bifunctional linkers,formation of a fusion polypeptide, and formation of biotin/streptavidinor biotin/avidin complexes by attaching either biotin orstreptavidin/avidin to the peptide and the complementary molecule to theBBB-transport facilitating compound.

Compounds which facilitate transport across the BBB include transferringreceptor binding antibodies (U.S. Pat. No. 5,527,527); certain lipoidalforms of dihydropyridine (see, e.g., U.S. Pat. No. 5,525,727); carrierpeptides, such as cationized albumin or Metenkephalin (and othersdisclosed in U.S. Pat. Nos. 5,442,043; 4,902,505; and 4,801,575);cationized antibodies (U.S. Pat. No. 5,004,697); and fatty acids such asdocosahexanoic acid (DHA; U.S. Pat. No. 4,933,324).

For other uses of the molecules to be transported, it may be preferredto administer the molecules in combination with a compound whichincreases transport of compounds across the blood-brain barrier (BBB).Such compounds, which need not be conjugated to a molecule fortransport, increase the transport of the molecule across the BBB intothe brain. A compound which increases transport across the BBB is one,for example, which increases the permeability of the BBB, preferablytransiently. Coadministration of a molecule for transport with such acompound permits the molecule to cross a permeabilized BBB. Examples ofsuch compounds include bradykinin and agonist derivatives (U.S. Pat. No.5,112,596); and receptor-mediated permeabilizers such as A-7 (U.S. Pat.No. 5,268,164 and 5,506,206).

According to a further aspect of the invention, pharmaceuticalcompositions containing the nucleic acid molecules, polypeptides, andbinding polypeptides of the invention are provided. The pharmaceuticalcompositions contain any of the foregoing therapeutic agents in apharmaceutically acceptable carrier. Thus, in a related aspect, theinvention provides a method for forming a medicament that involvesplacing a therapeutically effective amount of the therapeutic agent inthe pharmaceutically acceptable carrier to form one or more doses.

When administered, the therapeutic compositions of the present inventionare administered in pharmaceutically acceptable preparations. Suchpreparations may routinely contain pharmaceutically acceptableconcentrations of salt, buffering agents, preservatives, compatiblecarriers, supplementary immune potentiating agents such as adjuvants andcytokines and optionally other therapeutic agents.

The term “pharmaceutically acceptable” means a non-toxic material thatdoes not interfere with the effectiveness of the biological activity ofthe active ingredients. The term “physiologically acceptable” refers toa non-toxic material that is compatible with a biological system such asa cell, cell culture, tissue, or organism. The characteristics of thecarrier will depend on the route of administration. Physiologically andpharmaceutically acceptable carriers include diluents, fillers, salts,buffers, stabilizers, solubilizers, and other materials which are wellknown in the art.

The therapeutics of the invention can be administered by anyconventional route, including injection or by gradual infusion overtime. The administration may, for example, be oral, intravenous,intraperitoneal, intramuscular, intracavity, subcutaneous, ortransdermal. When antibodies are used therapeutically, a preferred routeof administration is by pulmonary aerosol. Techniques for preparingaerosol delivery systems containing antibodies are well known to thoseof skill in the art. Generally, such systems should utilize componentswhich will not significantly impair the biological properties of theantibodies, such as the paratope binding capacity (see, for example,Sciarra and Cutie, “Aerosols,” in Remington's Pharmaceutical Sciences,18th edition, 1990, pp 1694-1712). Those of skill in the art can readilydetermine the various parameters and conditions for producing antibodyaerosols without resort to undue experimentation. When using antisensepreparations of the invention, slow intravenous administration ispreferred. Preparations for administering therapeutics may also includecompounds for transport across the blood-brain barrier as describedelsewhere herein.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

The preparations of the invention are administered in effective amounts.An effective amount is that amount of a pharmaceutical preparation thatalone, or together with further doses, stimulates the desired response.In the case of treating a NR3B-associated motor neuron disorder, thedesired response is inhibiting the progression of the NR3B -associatedmotor neuron disorder. This may involve only slowing the progression ofthe disease temporarily, although more preferably, it involves haltingthe progression of the disease permanently. In the case of stimulatingan immune response, e.g. against a mutant NR3B polypeptide, the desiredresponse is an increase in antibodies or T lymphocytes which arespecific for the immunogen(s) employed. These responses can be monitoredby routine methods or can be monitored according to diagnostic methodsof the invention discussed herein.

The therapeutically effective amount of the NR3B molecule is that amounteffective to modulate NR3B functional activity levels and reduce,prevent, delay onset, or eliminate the NR3B-associated motor neurondisorder. For example, using the assays described in the Examplesection, testing can be performed to determine the NR3B functionalactivity in a subject's tissue and/or cells. Additional tests useful formonitoring the onset, progression, and/or remission, of NR3B-associatedmotor neuron disorders such as ALS, and other NR3B-associated motorneuron disorders known to one of ordinary skill in the art. As would beunderstood by one of ordinary skill, for some motor neuron disorders(e.g. reduced NR3B-function disorders) an effective amount would be theamount of NR3B molecules that increases NR3B functional activity to alevel that diminishes the disorder, as determined by the aforementionedtests. It is also understood that in other motor neuron disorders (e.g.increased function disorders) an effective amount would be that amountof NR3B molecules that decreases NR3B functional activity to a levelthat diminishes the disorder, as determined by the aforementioned tests.

The NR3B molecule dosage may be adjusted by the individual physician orveterinarian, particularly in the event of any complication. Atherapeutically effective amount typically varies from 0.01 mg/kg toabout 1000 mg/kg, preferably from about 0.1 mg/kg to about 200 mg/kg,and most preferably from about 0.2 mg/kg to about 20 mg/kg, in one ormore dose administrations daily, for one or more days.

According to another aspect of the invention, various diagnostic methodsare provided. In general, the methods are for diagnosing a motor neurondisorder characterized by aberrant expression of a NR3B molecule. Asused herein, “aberrant expression” is dependent upon the particularmotor neuron disorder (i.e., characterized by increased or decreasedNR3B molecule expression). Thus “aberrant expression refers to increasedexpression of normal NR3B, decreased expression (including noexpression) of a NR3B molecule (nucleic acid or polypeptide), orincreased expression of a “mutant NR3B molecule”. A mutant NR3B moleculerefers to a NR3B nucleic acid molecule which includes a mutation(deletion, addition, or substitution) or to a NR3B polypeptide molecule(e.g., gene product of mutant NR3B nucleic acid molecule) which includesa mutation, provided that the mutation results in a mutant NR3Bpolypeptide that has reduced or no non-mutant NR3B polypeptidefunctional activity. The diagnostic methods of the invention can be usedto detect the presence of a disorder associated with aberrant expressionof a NR3B molecule, as well as to assess the progression and/orregression of the disorder such as in response to treatment (e.g.,chemical therapy).

Expression of NR3B can be evaluated using standard methods known tothose of ordinary skill in the art. Such methods include, but are notlimited to: PCR, RT-PCT and antibody methods, which can be used toevaluate changes/alternations (for example, relative to normal) in theexpression of NR3B at the gene, transcript, and polypeptide levels,respectively. The above-mentioned methods can be used to evaluateexpression of NR3B molecule, whether or not it has normal functionalactivity. For example, a NR3B polypeptide maybe expressed but lacknormal function due to mutation that does not effect expression. Theabove-mentioned methods can be used to evaluate NR3B expressionregardless of functional activity.

Expression of NR3B can also be evaluated by determining the functionalactivity of NR3B in cells and tissues with methods including, but notlimited to, the electrophysiological activity assay described in theExamples. According to this aspect of the invention, the method fordiagnosing a disorder characterized by aberrant expression of a NR3Bmolecule involves: detecting in a first biological sample obtained froma subject, expression of a NR3B molecule, wherein decreased or increasedexpression of a NR3B molecule (depending upon the disorder as discussedherein) compared to a control sample indicates that the subject has amotor neuron disorder characterized by aberrant expression of a NR3Bmolecule.

As used herein, a “motor neuron disorder characterized by aberrantexpression of a NR3B molecule” refers to a motor neuron disorder inwhich there is a detectable difference in the expression and/or functionlevels of NR3B molecule(s) in selected cells of a subject compared tothe control levels of these molecules. Thus, a disorder characterized byaberrant expression of a NR3B molecule embraces overexpression of NR3B,underexpression (including no expression) of a NR3B molecule compared tocontrol levels of these molecules, as well as expression of a mutantNR3B nucleic acid molecule or mutant NR3B polypeptide. Such differencesin expression and/or function levels can be determined in accordancewith the diagnostic methods of the invention as disclosed herein.Disorders that are characterized by aberrant expression of a NR3Bmolecule include: motor neuron disorders associated with abnormal NMDAreceptor function, and/or receptor-ligand interaction. ExemplaryNR3B-associated motor neuron disorders include, but are not limited toALS.

In other motor neuron diseases and disorders, an increase in NR3Bexpression levels and/or functional activity would indicate the presenceof an “increased NR3B-activity disorder” in the subject. In addition,the expression of a mutant NR3B that has NR3B-like functional activity,can also indicate the presence of a motor neuron disorder, characterizedby an increase in NR3B expression (e.g. functional activity), asdescribed herein. Thus, depending upon the nature of the disorder, i.e.,whether it is attributable to reduced or elevated NR3B moleculeexpression, one skilled in the art can select the appropriate type oftreatment to modulate NR3B expression to achieve levels of thesemolecules which are found in individuals who are not diagnosed with suchmotor neuron disorders.

In certain embodiments, the methods of the invention are useful todiagnose a NR3B-associated motor neuron disorders including, but notlimited to ALS.

In yet other embodiments, the diagnostic methods are useful fordiagnosing the progression of a motor neuron disorder and evaluating itstreatment. According to these embodiments, the methods further involve:detecting in a second biological sample obtained from the subject,expression of a NR3B molecule, and comparing the expression of the NR3Bmolecule in the first biological sample and the second biologicalsample. In these embodiments, a decrease or an increase in theexpression of the NR3B molecule in the second biological sample comparedto the first biological sample indicates progression of the disorder.

In yet other embodiments, the diagnostic methods are useful fordiagnosing the regression of a motor neuron disorder. According to theseembodiments, the methods further involve: detecting in a secondbiological sample obtained from the subject, expression of a NR3Bmolecule, and comparing the expression of the NR3B molecule in the firstbiological sample and the second biological sample. In theseembodiments, an increase or a decrease in the expression of the NR3Bmolecule in the second biological sample compared to the firstbiological sample indicates regression of the disorder. As noted above,it is to be understood by one of ordinary skill that some disorders willbe characterized by an increase in NR3B functional activity and otherdisorders will be characterized by a decrease in NR3B functionalactivity.

In certain embodiments, the diagnostic methods of the invention detect aNR3B molecule that is a NR3B nucleic acid molecule as described above.In yet other embodiments, the methods involve detecting a NR3Bpolypeptide as described above.

Various detection methods can be used to practice the-diagnostic methodsof the invention. For example, the methods can involve contacting abiological sample with an agent that selectively binds to NR3B moleculesto detect these molecules. In certain embodiments, the NR3B molecule isa nucleic acid and the method involves using an agent that selectivelybinds to the NR3B molecule, e.g., a nucleic acid that hybridizes to SEQID NO: 1 under high stringency conditions. In yet other embodiments, theNR3B molecule is a polypeptide and the method involves using an agentthat selectively binds to the NR3B molecule, e.g., a bindingpolypeptide, such as an antibody, that selectively binds to SEQ ID NO:2.

According to still another aspect of the invention, kits for performingthe diagnostic methods of the invention are provided. The kits includenucleic acid-based kits or polypeptide-based kits (see FIG. 4).According to the former embodiment, the kits include: one or morenucleic acid molecules that hybridize to a NR3B nucleic acid moleculeunder high stringency conditions; and instructions for the use of thenucleic acid molecules in the diagnosis of a motor neuron disorderassociated with aberrant expression of a NR3B molecule. Nucleicacid-based kits optionally further include a first primer and a secondprimer, wherein the first primer and the second primer are constructedand arranged to selectively amplify at least a portion of an isolatedNR3B nucleic acid molecule comprising SEQ ID NO: 1. Alternatively,polypeptide based-kits are provided. Such kits include: one or morebinding polypeptides that selectively bind to a NR3B polypeptide andinstructions for the use of the binding polypeptides in the diagnosis ofa disorder associated with aberrant expression of a NR3B molecule. Inthe preferred embodiments, the binding polypeptides are antibodies orantigen-binding fragments thereof, such as those described above. Inthese and other embodiments, certain of the binding polypeptides bind tothe mutant NR3B polypeptide but do not bind to the non-mutant NR3Bpolypeptide to further distinguish the expression of these polypeptidesin a biological sample.

The foregoing kits can include instructions or other printed material onhow to use the various components of the kits for diagnostic purposes.

The biological sample can be located in vivo or in vitro. For example,the biological sample can be a tissue in vivo and the agent specific forthe NR3B nucleic acid molecule or polypeptide can be used to detect thepresence of such molecules in the tissue (e.g., for imaging portions ofthe tissue that express the NR3B gene products). Alternatively, thebiological sample can be located in vitro (e.g., a blood sample, biopsy(e.g., tumor or tissue biopsy), tissue extract). In a particularlypreferred embodiment, the biological sample can be a cell-containingsample, more preferably a sample containing neuronal cells. Samples oftissue and/or cells for use in the various methods described herein canbe obtained through standard methods. Samples can be surgical samples ofany type of tissue or body fluid. Samples can be used directly orprocessed to facilitate analysis (e.g., paraffin embedding). Exemplarysamples include a cell, a cell scraping, a cell extract, a blood sample,spinal fluid sample, a tissue biopsy, including punch biopsy, a tumorbiopsy, a bodily fluid, a tissue, or a tissue extract. Samples also canbe cultured receptors, cells, tissues, or organs.

NMDA receptors play beneficial roles in excitatory neurotransmission andtheir ligand-induced activation results in cation passage through thechannel. In some motor neuron disorders, such as ALS it may be desirableto increase NR3B expression and/or functional activity, such as byadministering a drug that increases NR3B activity in cells and tissuesof a subject. Such an increase in activity can be brought about by, forexample, increasing expression of NR3B, and/or increasing the level of amutant NR3B that has an increased level of activity as compared to theactivity level of normal NR3B.

In other motor neuron diseases and disorders, it may be desirable toreduce the activity of NR3B to increase cation passage through the NMDAreceptor channel. Cation passage maybe increased using methods such as:reducing expression of NR3B and/or inhibiting NR3B activity (e.g.competitive inhibition, binding inhibition). Increasing levels of amutant NR3B that is non-functional or reduced-function as compared tonormal NR3B, can also be therapeutically desirable in diseases anddisorders where a reduction in normal NR3B activity is desirable.

In general, the treatment methods involve administering an agent tomodulate expression of a NR3B molecule. In certain embodiments, themethod for treating a subject with a motor neuron disorder characterizedby aberrant expression of a NR3B molecule, involves administering to thesubject an effective amount of a NR3B nucleic acid molecule to treat thedisorder. In yet other embodiments, the method for treatment involvesadministering to the subject an effective amount of an antisensemolecule to modulate expression of a NR3B nucleic acid molecule andthereby, treat the motor neuron disorder. An exemplary molecule formodulating expression of a mutant NR3B nucleic acid molecule is anantisense molecule that is selective for the mutant nucleic acid andthat does not modulate expression of the non-mutant NR3B nucleic acidmolecule.

Alternatively, the method for treating a subject with a disordercharacterized by aberrant expression of a NR3B molecule involvesadministering to the subject an effective amount of a NR3B polypeptideto treat the disorder. In some embodiments, the treatment methodinvolves administering to the subject who has a disorder characterizedby hypoactivity of NMDA receptors, an effective amount of a bindingpolypeptide to inhibit a NR3B polypeptide, thereby increase activity ofthe NMDA receptor and treat the disorder. In other embodiments, thetreatment methods involve administering to the subject who has adisorder characterized by hyperactivity of NMDA receptors, an effectiveamount of a binding polypeptide to increase NR3B activity, therebydecreasing activity of the NMDA receptor. In certain preferredembodiments, the binding polypeptide is an antibody or anantigen-binding fragment thereof; more preferably, the antibodies orantigen-binding fragments are labeled with one or more cytotoxic agents

Some of the foregoing methods of the invention contemplate gene therapy.A procedure for performing ex vivo gene therapy is outlined in U.S. Pat.No. 5,399,346 and in exhibits submitted in the file history of thatpatent, all of which are publicly available documents. In general, itinvolves introduction in vitro of a functional copy of a gene into acell(s) of a subject which contains a defective copy of the gene, andreturning the genetically engineered cell(s) to the subject. Thefunctional copy of the gene is under operable control of regulatoryelements which permit expression of the gene in the geneticallyengineered cell(s). Numerous transfection and transduction techniques aswell as appropriate expression vectors are well known to those ofordinary skill in the art. In vivo gene therapy using vectors such asadenovirus, retroviruses, herpes virus, and targeted liposomes also iscontemplated according to the invention.

In preferred embodiments, a virus vector for delivering a nucleic acidmolecule encoding a NR3B polypeptide is selected from the groupconsisting of adenoviruses, adeno-associated viruses, poxvirusesincluding vaccinia viruses and attenuated poxviruses, Semliki Forestvirus, Venezuelan equine encephalitis virus, retroviruses, Sindbisvirus, and Ty virus-like particle. Examples of viruses and virus-likeparticles which have been used to deliver exogenous nucleic acidsinclude: replication-defective adenoviruses (e.g., Xiang et al.,Virology 219:220-227, 1996; Eloit et al., J. Virol. 7:5375-5381, 1997;Chengalvala et al., Vaccine 15:335-339, 1997), a modified retrovirus(Townsend et al., J. Virol. 71:3365-3374, 1997), a nonreplicatingretrovirus (Irwin et al., J. Virol. 68:5036-5044, 1994), a replicationdefective Semliki Forest virus (Zhao et al., Proc. Natl. Acad. Sci. USA92:3009-3013, 1995), canarypox virus and highly attenuated vacciniavirus-derivative (Paoletti, Proc. Natl. Acad. Sci. USA 93:11349-11353,1996), non-replicative vaccinia virus (Moss, Proc. Natl. Acad. Sci. USA93:11341-11348, 1996), replicative vaccinia virus (Moss, Dev. Biol.Stand. 82:55-63, 1994), Venzuelan equine encephalitis virus (Davis etal., J. Virol. 70:3781-3787, 1996), Sindbis virus (Pugachev et al.,Virology 212:587-594, 1995), and Ty virus-like particle (Allsopp et al.,Eur. J Immunol 26:1951-1959, 1996). In preferred embodiments, the virusvector is an adenovirus.

Another preferred virus for certain applications is the adeno-associatedvirus, a double-stranded DNA virus. The adeno-associated virus iscapable of infecting a wide range of cell types and species and can beengineered to be replication-deficient. It further has advantages, suchas heat and lipid solvent stability, high transduction frequencies incells of diverse lineages, including hematopoietic cells, and lack ofsuperinfection inhibition thus allowing multiple series oftransductions. The adeno-associated virus can integrate into humancellular DNA in a site-specific manner, thereby minimizing thepossibility of insertional mutagenesis and variability of inserted geneexpression. In addition, wild-type adeno-associated virus infectionshave been followed in tissue culture for greater than 100 passages inthe absence of selective pressure, implying that the adeno-associatedvirus genomic integration is a relatively stable event. Theadeno-associated virus can also function in an extrachromosomal fashion.

In general, other preferred viral vectors are based on non-cytopathiceukaryotic viruses in which non-essential genes have been replaced withthe gene of interest. Non-cytopathic viruses include retroviruses, thelife cycle of which involves reverse transcription of genomic viral RNAinto DNA with subsequent proviral integration into host cellular DNA.Adenoviruses and retroviruses have been approved for human gene therapytrials. In general, the retroviruses are replication-deficient (i.e.,capable of directing synthesis of the desired polypeptides, butincapable of manufacturing an infectious particle). Such geneticallyaltered retroviral expression vectors have general utility for thehigh-efficiency transduction of genes in vivo. Standard protocols forproducing replication-deficient retroviruses (including the steps ofincorporation of exogenous genetic material into a plasmid, transfectionof a packaging cell lined with plasmid, production of recombinantretroviruses by the packaging cell line, collection of viral particlesfrom tissue culture media, and infection of the target cells with viralparticles) are provided in Kriegler, M., “Gene Transfer and Expression,A Laboratory Manual,” W. H. Freeman Co., New York (1990) and Murry, E.J. Ed. “Methods in Molecular Biology,” vol. 7, Humana Press, Inc.,Cliffton, N.J. (1991).

Preferably the foregoing nucleic acid delivery vectors: (1) containexogenous genetic material that can be transcribed and translated in amammalian cell and that can suppress NR3B-associated disease (e.g. motorneuron-associated disorders), and preferably (2) contain on a surface aligand that selectively binds to a receptor on the surface of a targetcell, such as a mammalian cell, and thereby gains entry to the targetcell.

Various techniques may be employed for introducing nucleic acidmolecules of the invention into cells, depending on whether the nucleicacid molecules are introduced in vitro or in vivo in a host. Suchtechniques include transfection of nucleic acid molecule-CaPO₄precipitates, transfection of nucleic acid molecules associated withDEAE, transfection or infection with the foregoing viruses including thenucleic acid molecule of interest, liposome-mediated transfection, andthe like. For certain uses, it is preferred to target the nucleic acidmolecule to particular cells. In such instances, a vehicle used fordelivering a nucleic acid molecule of the invention into a cell (e.g., aretrovirus, or other virus; a liposome) can have a targeting moleculeattached thereto. For example, a molecule such as an antibody specificfor a surface membrane polypeptide on the target cell or a ligand for areceptor on the target cell can be bound to or incorporated within thenucleic acid molecule delivery vehicle. Especially preferred aremonoclonal antibodies. Where liposomes are employed to deliver thenucleic acid molecules of the invention, proteins that bind to a surfacemembrane protein associated with endocytosis may be incorporated intothe liposome formulation for targeting and/or to facilitate uptake. Suchproteins include capsid proteins or fragments thereof tropic for aparticular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half life, and the like.Polymeric delivery systems also have been used successfully to delivernucleic acid molecules into cells, as is known by those skilled in theart. Such systems even permit oral delivery of nucleic acid molecules.

In addition to delivery through the use of vectors, NR3B nucleic acidsmay be delivered to cells without vectors, e.g. as “naked” nucleic aciddelivery using methods known to those of skill in the art.

Various forms of the NR3B polypeptide or nucleic acid, as describedherein, can be administered and delivered to a mammalian cell (e.g., byvirus or liposomes, or by any other suitable methods known in the art orlater developed). The method of delivery can be modified to targetcertain cells, and in particular, cell surface receptor molecules orantigens present on neuronal cells. Methods of targeting cells todeliver nucleic acid constructs are known in the art. The NR3Bpolypeptide can also be delivered into cells by expressing a recombinantprotein fused with peptide carrier molecules, examples of which, thoughnot intended to be limiting, are tat or antennapedia. These deliverymethods are known to those of skill in the art and are described in U.S.Pat. No. 6,080,724, and U.S. Pat. No. 5,783,662, the entire contents ofwhich are hereby incorporated by reference.

In addition to the methods described herein for delivering exogenousNR3B, expression of endogenous normal or mutant NR3B can be induced(e.g., upregulated) by the administration of chemicals or othermolecules that specifically increase the level of NR3B mRNA and/orpolypeptide. Such induction and/or upregulation of endogenous NR3B mayoccur through methods that include, but not limited to: (a) activationof the NR3B promoter, (b) stabilization of NR3B mRNA, (c) increasedtranslation of NR3B polypeptide and (d) stabilization of NR3Bpolypeptide.

The invention further provides efficient methods of identifyingpharmacological agents or lead compounds for agents which mimic thefunctional activity of a NR3B molecule. Such NR3B functional activitiesinclude excitatory neurotransmission and modulation of cation passage.Generally, the screening methods involve assaying for compounds whichmodulate (up- or down-regulate) a NR3B functional activity.

The DNA, mRNA, vectors, receptor subunits, receptor subunit combinationsand cells provided herein permit production of selected NMDA receptorsubunits and specific combinations thereof, as well as antibodies tosaid receptor subunits. This provides a means to prepare synthetic orrecombinant receptors and receptor subunits that are substantially freeof contamination from many other receptor polypeptides whose presencecan interfere with analysis of a single NMDA receptor subtype. Theavailability of desired receptor subtypes makes it possible to observethe effect of a drug substance on a particular receptor subtype orcombination of NMDA receptor subunits, and to thereby perform initial invitro screening of the drug substance in a test system that is specificfor humans and specific for a human NMDA receptor subtype or combinationof NMDA receptor subunits, including NR3B subunits. The availability ofspecific antibodies makes it possible to identify the subunitcombinations expressed in vivo. Such specific combinations can then beemployed as preferred targets in drug screening.

The ability to screen drug substances in vitro to determine the effectof the drug on specific receptor compositions should permit thedevelopment and screening of receptor subtype-specific ordisease-specific drugs. Also, testing of single receptor subunits orspecific combinations of various types of receptor subunits with avariety of potential agonists or antagonists provides additionalinformation with respect to the function and activity of the individualsubunits and should lead to the identification and design of compoundsthat are capable of specific interaction with one or more types ofreceptor subunits (e.g. NR3B) or receptor subtypes. The resulting drugsshould exhibit fewer unwanted side effects than drugs identified byscreening with cells that express a variety of receptor subtypes.

The invention also provides methods for identifying compounds that bindto human N-methyl-D-aspartate (NMDA) receptor subunit(s) (such as NR3B).For example, NR3B polypeptides can be used in a competitive bindingassay. Such an assay can accommodate the rapid screening of a largenumber of compounds to determine which compounds, if any, are capable ofbinding to NMDA receptors. Subsequently, more detailed assays can becarried out with those compounds found to bind, to further determinewhether such compounds act as modulators, agonists, or antagonists ofinvention receptors.

Another application of the binding assay of the invention is the assayof test samples (e.g., biological fluids) for the presence or absence ofreceptors of the present invention. Thus, for example, spinal fluid froma patient displaying symptoms related to glutamatergic pathwaydysfunction can be assayed to determine if the observed symptoms areperhaps caused by over- or under-production of receptor subunits, suchas NR3B.

The binding assays contemplated by the present invention can be carriedout in a variety of ways, as can readily be identified by those of skillin the art. For example, competitive binding assays can be employed,such as radioreceptor assays, fluorescence assays, and the like.

In accordance with a further embodiment of the present invention, thereis provided a bioassay for identifying compounds which modulate theactivity of NMDA receptors of the invention. An example of such abioassay includes: exposing cells containing DNA encoding NMDA receptorsubunit(s), wherein the cells express functional NMDA receptors, to oneor more compounds whose ability to modulate the ion channel activity ofthe receptors is sought to be determined; and monitoring the cells forchanges in ion channel activity.

The above-described bioassay can be used to identify agonists andantagonists for human NMDA receptors. According to this method,recombinant NMDA receptors are contacted with an “unknown” or testsubstance (in the further presence of a known NMDA agonist, whenantagonist activity is being tested), the ion channel activity of theknown glutamate receptor is monitored subsequent to the contact with the“unknown” or test substance, and those substances which increase ordecrease the ion channel response of the known glutamate receptor(s) areidentified as functional ligands (i.e., modulators, agonists orantagonists) for human NMDA receptors. (See electrophysiological assayin Example Section).

In accordance with a particular embodiment of the present invention,recombinant NMDA receptor NR3B-expressing mammalian cells or oocytes canbe contacted with a test compound, and the modulating effect(s) thereofcan then be evaluated by comparing the NMDA receptor-mediated responsein the presence and absence of test compound, or by comparing theresponse of test cells, or control cells (i.e., cells that do notexpress NMDA receptor NR3B subunits, or express a normal level offunctional NR3B subunits), to the presence of the compound. Arecombinant NMDA receptor of the invention includes NR3B subunits andits response to the test compounds may be compared to responses of cellswith NMDA receptors lacking NR3B subunits, or containing mutant NR3Bsubunits.

In accordance with yet another embodiment of the present invention, theion channel activity of N-methyl-D-aspartate (NMDA) receptors containingNR3B can be modulated by contacting such receptors with an effectiveamount of at least one compound identified by the above-describedbioassay.

A wide variety of assays for pharmacological agents can be used inaccordance with this aspect of the invention, including, labeled invitro protein-protein binding assays, electrophoretic mobility shiftassays, immunoassays, cell-based assays such as two- or three-hybridscreens, expression assays, etc. The assay mixture comprises a candidatepharmacological agent. Typically, a plurality of assay mixtures are runin parallel with different agent concentrations to obtain a differentresponse to the various concentrations. Typically, one of theseconcentrations serves as a negative control, i.e., at zero concentrationof agent or at a concentration of agent below the limits of assaydetection. Candidate agents encompass numerous chemical classes,although typically they are organic compounds. Preferably, the candidatepharmacological agents are small organic compounds, i.e., those having amolecular weight of more than 50 yet less than about 2500, preferablyless than about 1000 and, more preferably, less than about 500.Candidate agents comprise functional chemical groups necessary forstructural interactions with polypeptides and/or nucleic acid molecules,and typically include at least an amine, carbonyl, hydroxyl or carboxylgroup, preferably at least two of the functional chemical groups andmore preferably at least three of the functional chemical groups. Thecandidate agents can comprise cyclic carbon or heterocyclic structureand/or aromatic or polyaromatic structures substituted with one or moreof the above-identified functional groups. Candidate agents also can bebiomolecules such as peptides, saccharides, fatty acids, sterols,isoprenoids, purines, pyrimidines, derivatives or structural analogs ofthe above, or combinations thereof and the like. Where the agent is anucleic acid molecule, the agent typically is a DNA or RNA molecule,although modified nucleic acid molecules as defined herein are alsocontemplated.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides, synthetic organic combinatorial libraries, phagedisplay libraries of random peptides, and the like. Alternatively,libraries of natural compounds in the form of bacterial, fungal, plantand animal extracts are available or readily produced. Additionally,natural and synthetically produced libraries and compounds can bereadily be modified through conventional chemical, physical, andbiochemical means. Further, known pharmacological agents may besubjected to directed or random chemical modifications such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs of the agents.

A variety of other reagents also can be included in the mixture. Theseinclude reagents such as salts, buffers, neutral proteins (e.g.,albumin), detergents, etc. which may be used to facilitate optimalprotein-protein and/or protein-nucleic acid binding. Such a reagent mayalso reduce non-specific or background interactions of the reactioncomponents. Other reagents that improve the efficiency of the assay suchas protease inhibitors, nuclease inhibitors, antimicrobial agents, andthe like may also be used.

An exemplary binding assay is described herein. In general the mixtureof the foregoing assay materials is incubated under conditions whereby,but for the presence of the candidate pharmacological agent, the NR3Bmolecule specifically binds the binding agent (e.g., ligand). The orderof addition of components, incubation temperature, time of incubation,and other parameters of the assay may be readily determined. Suchexperimentation merely involves optimization of the assay parameters,not the fundamental composition of the assay. Incubation temperaturestypically are between 4° C. and 40° C. Incubation times preferably areminimized to facilitate rapid, high throughput screening, and typicallyare between 0.1 and 10 hours.

After incubation, the presence or absence of specific binding betweenthe NR3B molecule and one or more binding agents is detected by anyconvenient method available to the user. For cell-free binding typeassays, a separation step is often used to separate bound from unboundcomponents. The separation step may be accomplished in a variety ofways. Conveniently, at least one of the components is immobilized on asolid substrate, from which the unbound components may be easilyseparated. The solid substrate can be made of a wide variety ofmaterials and in a wide variety of shapes, e.g., microtiter plate,microbead, dipstick, resin particle, etc. The substrate preferably ischosen to maximum signal to noise ratios, primarily to minimizebackground binding, as well as for ease of separation and cost.

Separation may be effected for example, by removing a bead or dipstickfrom a reservoir, emptying or diluting a reservoir such as a microfiterplate well, rinsing a bead, particle, chromotograpic column or filterwith a wash solution or solvent. The separation step preferably includesmultiple rinses or washes. For example, when the solid substrate is amicrotiter plate, the wells may be washed several times with a washingsolution, which typically includes those components of the incubationmixture that do not participate in specific bindings such as salts,buffer, detergent, non-specific protein, etc. Where the solid substrateis a magnetic bead, the beads may be washed one or more times with awashing solution and isolated using a magnet.

Detection may be effected in any convenient way for cell-based assayssuch as two- or three-hybrid screens. For cell-free binding assays, oneof the components usually comprises, or is coupled to, a detectablelabel. A wide variety of labels can be used, such as those that providedirect detection (e.g., radioactivity, luminescence, optical or electrondensity, etc.) or indirect detection (e.g., epitope tag such as the FLAGepitope, enzyme tag such as horseseradish peroxidase, etc.). The labelmay be bound to a NR3B binding partner (e.g., polypeptide), orincorporated into the structure of the binding partner.

A variety of methods may be used to detect the label, depending on thenature of the label and other assay components. For example, the labelmay be detected while bound to the solid substrate or subsequent toseparation from the solid substrate. Labels may be directly detectedthrough optical or electron density, radioactive emissions, nonradiativeenergy transfers, etc. or indirectly detected with antibody conjugates,strepavidin-biotin conjugates, etc. Methods for detecting the labels arewell known in the art.

Detection may be effected in any convenient way for cell-based assayssuch as a calcium influx assay. The calcium influx resulting fromactivation of the NMDA receptor by ligand binding typically alters adirectly or indirectly detectable product, e.g., a calcium sensitivemolecule such as fura-2-AM. For cell free binding assays, one of thecomponents usually comprises, or is coupled to, a detectable label. Awide variety of labels can be used, such as those that provide directdetection (e.g., radioactivity, luminescence, optical or electrondensity, etc). or indirect detection (e.g., epitope tag such as the FLAGepitope, enzyme tag such as horseradish peroxidase, etc.). The label maybe bound to a NR3B polypeptide, decoy peptide, or the candidatepharmacological agent.

A variety of methods may be used to detect the label, depending on thenature of the label and other assay components. For example, the labelmay be detected while bound to the solid substrate or subsequent toseparation from the solid substrate. Labels may be directly detectedthrough optical or electron density, radioactive emissions, nonradiativeenergy transfers, etc. or indirectly detected with antibody conjugates,streptavidin-biotin conjugates, etc. Methods for detecting the labelsare well known in the art.

Thus the present invention includes automated drug screening assays foridentifying compositions having the ability to inhibit ion influx in acell induced by ligand binding, thus contributing to a detectable changein the cytoplasmic level of a predetermined ion in the cell, thecytoplasm of which cell contains an indicator which is sensitive to theion. The method is carried out in an apparatus which is capable ofdelivering a reagent solution to a plurality of predeterminedcell-containing compartments of a vessel and measuring the detectablechange in the cytoplasmic level of the ion in the cells of thepredetermined compartments, such as the apparatus and method describedin U.S. Pat. No. 6,057,114. Exemplary methods include the followingsteps. First, a divided culture vessel is provided that has one or morecompartments which contain viable cells which, when exposed to NMDAagonists, have a detectable change in the concentration of thepredetermined ion in the cytoplasm. The cytoplasms of the cells includean amount of an ion-sensitive fluorescent indicator sufficient to detecta change, if any, in the concentration of the predetermined ion. NMDAagonists are added to the cells to induce calcium influx and/ordepolarization. Next, one or more predetermined cell-containingcompartments are aligned with a predetermined position (e.g., alignedwith a fluid outlet of an automatic pipette) and an aliquot of asolution containing a compound or mixture of compounds being tested forits ability to modulate NR3B-mediated calcium influx and/ordepolarization is delivered to the predetermined compartment(s) with anautomatic pipette. Finally, fluorescence emitted by the ion-sensitiveindicator in response to an excitation wavelength is measured for apredetermined amount of time, preferably by aligning saidcell-containing compartment with a fluorescence detector. Preferably,fluorescence also measured prior to adding NMDA receptor agonist to thecells and/or prior to adding the compound to the wells, to establishe.g., background and/or baseline values for fluorescence.

In accordance with the various assays of the present invention, cellsare employed which have ion channels and/or receptors, the activation ofwhich by NMDA agonists results in a change in the level of a cation oranion in the cytoplasm. The cytoplasm of the cells employed are loadedwith a fluorescent indicator which is sufficiently sensitive to saidion. By the phrase “sufficiently sensitive fluorescent indicator” ismeant a fluorescent compound which, in the presence of, and over a rangeof physiological concentrations of, a particular ion, is capable ofproducing distinguishable levels of fluorescence intensity. Preferably,a fluorescent indicator should be able to produce detectably differentintensities of fluorescence in response to relatively small changes inion concentration. The relative intensities of fluorescence when thereceptors or ion channels have not been activated, as compared to whenthe receptors or ion channels have been activated, preferably differ byat least about 50% or more, more preferably by at least about 100-200%.

Any cell which is capable, upon exposure to NMDA agonist, of directlyincreasing the intracellular concentration of calcium, such as bypermitting calcium influx through the NMDA receptor channels, or bycausing release of calcium from intracellular stores, may be used in theassay. Preferably neuronal cell lines or cultured neurons are used.

Activation of cellular receptors and/or ion channels (e.g., NMDA-typechannels) by incubation with NMDA agonists, may result in a transientincrease in the level of intracellular calcium (and/or other ions). Theinitial increase in calcium may be detected as a rapid increase influorescence (e.g., within one to two seconds) after the addition of theNMDA agonist. As shown herein, calcium influx is generally short-lived,but depolarization is longer lasting. Fluorescence levels in thecytoplasm resulting from calcium influx typically increase to a peakvalue and then typically decline as excess calcium ions are removed bynormal cellular mechanisms. The speed at which the fluorescence can beanalyzed is important for analysis of the kinetics of the reaction, ifit is desired to measure kinetics.

The cells used in the assays of the invention are loaded with afluorescent indicator which is sufficiently sensitive so as to producedetectable changes in fluorescence intensity in response to changes inthe concentration of the ions in the cytoplasm. It is particularlypreferred to use a fluorescent indicator which has such sensitivity inthe presence of calcium ions. Among the fluorescent indicators which maybe employed are the following compounds commercially available from,e.g., Molecular Probes, Inc., Eugene Oreg.: DiBAC₄(3) (B-438), Quin-2(AM Q-1288), Fura-2 (AM F-1225), Indo-1 (AM I-1226), Fura-3 (AM F-1228),Fluo-3 (AM F-1241), Rhod-2, (AM R-1244), BAPTA (AM B-1205),5,5′-dimethyl BAPTA (AM D-1207), 4,4′-difluoro BAPTA (AM D-1216),5,5′-difluoro BAPTA (AM D-1209), 5,5′-dibromo BAPTA (AM D-1213), CalciumGreen (C-3011), Calcium Orange (C-3014), Calcium Crimson (C-3017),Fura-5 (F-3023), Fura-Red (F-3020), SBFI (S-1262), PBFI (P-1265),Mag-Fura-2 (AM M-1291), Mag-Indo-1 (AM M-1294), Mag-Quin-2 (AM M-1299),Mag-Quin-1 (AM M-1297), SPQ (M-440), and SPA (S-460).

It is contemplated that each of the individual wells contain the samecell type so that multiple compounds (obtained from different reagentsources in the apparatus or contained within different wells) can bescreened and compared for modulating activity with respect to NMDAagonist induction of calcium influx and/or depolarization.

In another of its aspects the invention entails automated antagonistassays. Antagonist assays, including drug screening assays, may becarried out by incubating the cells (e.g., neurons) with NMDA agoniststo induce calcium influx and/or depolarization, in the presence andabsence of one or more compounds added to the solution bathing the cellsin the respective wells of the microtiter plate for an amount of timesufficient for the compound(s) to modulate calcium influx and/ordepolarization, and measuring the level of fluorescence in the cells ascompared to the level of fluorescence in either the same cell, orsubstantially identical cell, in the absence of the NMDA agonist. Suchassays can be used to identify compounds/chemicals that increase ordecrease the function of NR3B subunits in the NMDA receptors. Asdescribed above, for some disorders, it may be desirable to identifycompounds to increase the activity of the NR3B subunit and in otherdisorders it may be desirable identify compounds to decrease theactivity of the NR3B subunit.

As will be understood by the person of ordinary skill in the art,compounds exhibiting agonist or antagonist activity in an assay ofcalcium influx or depolarization will either increase or decreaseintracellular ion levels (agonist) or inhibit (antagonist) an increaseor decrease in the intracellular concentration of ions after incubationof cells with NMDA agonist. It is desirable to measure the amount ofagonist or antagonist activity in a linear range of the assay system,such that small but significant increases or decreases in fluorescencerelative to control well (e.g., devoid of the test compound) may beobserved. It is well within the skill of the art to determine a volumeand concentration of a reagent solution which causes a suitableactivation response in cells so that modulation of the calcium influxand/or depolarization may be reliably detected.

At a suitable time after addition of the NMDA agonist to initiatecalcium influx and/or depolarization, the plate is moved, if necessary,so that the cell-containing assay well is positioned for measurement offluorescence emission. Because a change in the fluorescence signal maybegin within the first few seconds after addition of test compounds, itis desirable to align the assay well with the fluorescence readingdevice as quickly as possible, with times of about two seconds or lessbeing desirable. In preferred embodiments of the invention, where theapparatus is configured for detection through the bottom of the well(s)and compounds are added from above the well(s), fluorescence readingsmay be taken substantially continuously, since the plate does not needto be moved for addition of reagent. The well and fluorescence-readingdevice should remain aligned for a predetermined period of time suitableto measure and record the change in intracellular ion, e.g., calcium,concentration. In preferred embodiments of the invention thefluorescence after activation is read and recorded until thefluorescence change is maximal and then begins to reduce. An empiricallydetermined time period may be chosen which covers the transient rise andfall (or fall and rise) of intracellular ion levels in response toaddition of the compound. When the apparatus is configured to detectfluorescence from above the plate, it is preferred that the bottom ofthe wells are colored black to reduce the background fluorescence andthereby decreases the noise level in the fluorescence reader.

After finishing reading and recording the fluorescence in one well, thejust described apparatus steps are repeated with the next well(s) in theseries so as to measure pre-reagent fluorescence, add reagent andmeasure and record the transient change, if any, in fluorescence. Theapparatus of the present invention is programmable to begin the steps ofan assay sequence in a predetermined first well (or row or column ofwells) and proceed sequentially down the columns and across the rows ofthe plate in a predetermined route through well number n.

In assays of cells treated with NMDA agonist to cause an increase inintracellular calcium ion concentration and/or depolarization, it ispreferred that the fluorescence data from replicate wells of cellstreated with the same compound are collected and recorded (e.g., storedin the memory of a computer) for calculation of fluorescence and/orintracellular calcium ion concentration.

In assays of compounds that inhibit calcium influx and/ordepolarization, the results can be expressed as a percentage of themaximal response caused by NMDA agonist. The maximal fluorescenceincrease caused by NMDA agonist is defined as being 100% response. Forcompounds effective for reducing calcium influx and/or depolarizationinduced by NMDA agonist, the maximal fluorescence recorded afteraddition of a compound to wells containing NMDA agonist is detectablylower than the fluorescence recorded in the presence of only NMDAagonist.

The fluorescence indicator-based assays of the present invention arethus useful for rapidly screening compounds to identify those thatmodulate calcium influx and/or depolarization that ultimately results inan altered concentration of ions in the cytoplasm of a cell. Forexample, the assays can be used to determine functional activity of NR3Bsubunits upon application of NMDA agonist and can be used to compareactivity of NMDA receptors with normal NR3B subunit and NMDA receptorswith variant (mutant) NR3B subunits. The assays can preferably be usedto determine the effect on functional activity of NR3B subunitcontaining NMDA receptors, for both normal and variant (mutant) NR3Bsubunits.

Automation of the fluorescent dye-based assays of the invention can beperformed as described in U.S. Pat. No. 6, 057,114. Automation canprovide increased efficiency in conducting the assays and increasedreliability of the results by permitting multiple measurements overtime, thus also facilitating determination of the kinetics of thecalcium influx or depolarization effects.

For example, to accomplish rapid compound addition and rapid reading ofthe fluorescence response, the fluorometer can be modified by fitting anautomatic pipetter and developing a software program to accomplishprecise computer control over both the fluorometer and the automaticpipetter. By integrating the combination of the fluorometer and theautomatic pipetter and using a microcomputer to control the commands tothe fluorometer and automatic pipetter, the delay time between reagentaddition and fluorescence reading can be significantly reduced.Moreover, both greater reproducibility and higher signal-to-noise ratioscan be achieved as compared to manual addition of reagent because thecomputer repeats the process precisely time after time. Moreover, thisarrangement permits a plurality of assays to be conducted concurrentlywithout operator intervention. Thus, with automatic delivery of reagentfollowed by multiple fluorescence measurements, reliability of thefluorescent dye-based assays as well as the number of assays that can beperformed per day are advantageously increased.

The invention further includes nucleic acid or protein microarrays withNR3B polypeptides or nucleic acids encoding such polypeptides. In thisaspect of the invention, standard techniques of microarray technologyare utilized to assess expression of the NR3B polypeptides and/oridentify biological constituents that bind such polypeptides. Proteinmicroarray technology, which is also known by other names including:protein chip technology and solid-phase protein array technology, iswell known to those of ordinary skill in the art and is based on, butnot limited to, obtaining an array of identified polypeptides orproteins on a fixed substrate, binding target molecules or biologicalconstituents to the polypeptides, and evaluating such binding. See,e.g., G. MacBeath and S. L. Schreiber, Printing Proteins as Microarraysfor High-Throughput Function Determination,” Science289(5485):1760-1763, 2000. Protein arrays, particularly arrays that bindNR3B polypeptides, also can be used for diagnostic applications, such asfor identifying subjects that have a condition characterized by NR3Bpolypeptide expression.

Microarray substrates include but are not limited to glass, silica,aluminosilicates, borosilicates, metal oxides such as alumina and nickeloxide, various clays, nitrocellulose, or nylon. The microarraysubstrates may be coated with a compound to enhance synthesis of a probe(peptide or nucleic acid) on the substrate. Coupling agents or groups onthe substrate can be used to covalently link the first nucleotide oramino acid to the substrate. A variety of coupling agents or groups areknown to those of skill in the art. Peptide or nucleic acid probes thuscan be synthesized directly on the substrate in a predetermined grid.Alternatively, peptide or nucleic acid probes can be spotted on thesubstrate, and in such cases the substrate may be coated with a compoundto enhance binding of the probe to the substrate. In these embodiments,presynthesized probes are applied to the substrate in a precise,predetermined volume and grid pattern, preferably utilizing acomputer-controlled robot to apply probe to the substrate in acontact-printing manner or in a non-contact manner such as inkjet orpiezo-electric delivery. Probes may be covalently linked to thesubstrate.

Targets are polypeptides or proteins and may be natural or synthetic.The tissue may be obtained from a subject or may be grown in culture(e.g. from a cell line).

In some embodiments of the invention, one or more control polypeptide orprotein molecules are attached to the substrate. Preferably, controlpolypeptide or protein molecules allow determination of factors such aspolypeptide or protein quality and binding characteristics, reagentquality and effectiveness, and analysis thresholds and success.

Nucleic acid microarray technology, which is also known by other namesincluding: DNA chip technology, gene chip technology, and solid-phasenucleic acid array technology, is well known to those of ordinary skillin the art and is based on, but not limited to, obtaining an array ofidentified nucleic acid probes on a fixed substrate, labeling targetmolecules with reporter molecules (e.g., radioactive, chemiluminescent,or fluorescent tags such as fluorescein, Cye3-dUTP, or CyeS-dUTP),hybridizing target nucleic acids to the probes, and evaluatingtarget-probe hybridization. A probe with a nucleic acid sequence thatperfectly matches the target sequence will, in general, result indetection of a stronger reporter-molecule signal than will probes withless perfect matches. Many components and techniques utilized in nucleicacid microarray technology are presented in The Chipping Forecast,Nature Genetics, Vol.21, January 1999, the entire contents of which isincorporated by reference herein.

According to the present invention, nucleic acid microarray substratesmay include but are not limited to glass, silica, aluminosilicates,borosilicates, metal oxides such as alumina and nickel oxide, variousclays, nitrocellulose, or nylon. In all embodiments, a glass substrateis preferred. According to the invention, probes are selected from thegroup of nucleic acids including, but not limited to: DNA, genomic DNA,cDNA, and oligonucleotides; and may be natural or synthetic.Oligonucleotide probes preferably are 20 to 25-mer oligonucleotides andDNA/cDNA probes preferably are 500 to 5000 bases in length, althoughother lengths may be used. Appropriate probe length may be determined byone of ordinary skill in the art by following art-known procedures. Inone embodiment, preferred probes are sets of one or more of the NR3Bnucleic acid molecules set forth herein. Probes may be purified toremove contaminants using standard methods known to those of ordinaryskill in the art such as gel filtration or precipitation.

In one embodiment, the microarray substrate may be coated with acompound to enhance synthesis of the probe on the substrate. Suchcompounds include, but are not limited to, oligoethylene glycols. Inanother embodiment, coupling agents or groups on the substrate can beused to covalently link the first nucleotide or olignucleotide to thesubstrate. These agents or groups may include, for example, amino,hydroxy, bromo, and carboxy groups. These reactive groups are preferablyattached to the substrate through a hydrocarbyl radical such as analkylene or phenylene divalent radical, one valence position occupied bythe chain bonding and the remaining attached to the reactive groups.These hydrocarbyl-groups may contain up to about ten carbon atoms,preferably up to about six carbon atoms. Alkylene radicals are usuallypreferred containing two to four carbon atoms in the principal chain.These and additional details of the process are disclosed, for example,in U.S. Pat. No. 4,458,066, which is incorporated by reference in itsentirety.

In one embodiment, probes are synthesized directly on the substrate in apredetermined grid pattern using methods such as light-directed chemicalsynthesis, photochemical deprotection, or delivery of nucleotideprecursors to the substrate and subsequent probe production.

In another embodiment, the substrate may be coated with a compound toenhance binding of the probe to the substrate. Such compounds include,but are not limited to: polylysine, amino silanes, amino-reactivesilanes (Chipping Forecast, 1999) or chromium. In this embodiment,presynthesized probes are applied to the substrate in a precise,predetermined volume and grid pattern, utilizing a computer-controlledrobot to apply probe to the substrate in a contact-printing manner or ina non-contact manner such as ink jet or piezo-electric delivery. Probesmay be covalently linked to the substrate with methods that include, butare not limited to, UV-irradiation. In another embodiment probes arelinked to the substrate with heat.

Targets for microarrays are nucleic acids selected from the group,including but not limited to: DNA, genomic DNA, cDNA, RNA, mRNA and maybe natural or synthetic. In all embodiments, nucleic acid targetmolecules from human tissue are preferred. The tissue may be obtainedfrom a subject or may be grown in culture (e.g. from a cell line). Insome embodiments, targets for microarrays are proteins/peptides.

In embodiments of the invention one or more control nucleic acidmolecules are attached to the substrate. Preferably, control nucleicacid molecules allow determination of factors such as nucleic acidquality and binding characteristics, reagent quality and effectiveness,hybridization success, and analysis thresholds and success. Controlnucleic acids may include but are not limited to expression products ofgenes such as housekeeping genes or fragments thereof.

The invention will be more fully understood by reference to thefollowing examples. These examples, however, are merely intended toillustrate the embodiments of the invention and are not to be construedto limit the scope of the invention.

EXAMPLES Example 1 Identification of a Novel Glutamate Receptor

Introduction

A TBLASTN search was performed on the published human genomic sequenceusing the amino acid sequence of the mouse δ2 subunit as a querysequence. This search detected a stretch of genomic sequence withsignificant homology to members of the glutamate receptor family on acontig from human chromosome 19p13.3 (accession AC004528). This sequencewas then used to search the htgs database (unnished high throughputgenomic sequences including phases 0, 1 and 2) and the search identifiedthe mouse homologue (AC087114 and AC073805) on chromosome 10. A searchof the expressed sequence tag (ESI) database with these sequencesidentified the following ESTs: accession AL040053 human); AW045848,BE864387, AW048083, BE955769 (mouse); AW525909, BE108608, BE112464(rat). AW045848 and BE864387 are the same clone sequenced from oppositeends. This clone, which corresponds to the extracellular domain of theputative receptor, was used to screen a mouse spinal cord cDNA libraryand for in situ hybridization (Clontech, Palo Alto, Calif.).

RT-PCR and in situ Hybridization

RT-PCR was performed with primers spanning predicted exons 1(CCTCTATAACCTTTCCCGAGG) (SEQ ID NO: 11) and 2 (CTAGAGCAATGTCCTCCCAGG)(SEQ ID NO: 12) of mouse NR3B. The primer set NR1 (5′ primer:GATCCTCGAGCCATGGAGATCGCCTACAAGCGACAC (SEQ ID NO:13), 3′ primer:GATCGGATCCGCATGCTCAGCTCTCCCTATGACGGG (SEQ ID NO: 14) was used as apositive control for the RT-PCR. For in situ hybridizations on brain andspinal cord sections from male mice (C57/BL6, 6 weeks), either ³³P ordigoxigenin-labeled RNA probes were used (Simmons et al., 1989; Lu etal., in press). Serotonin was immunohistochemically detected withanti-serotonin antibody (Incstar, Stillwater, Minn.).

Electrophysiology in HEK293

To express the receptors in HEK293 cells, the cells were transfectedwith 1 μg of each plasmid unless otherwise stated (Shi et al., 1999).The NR1 and NR2A were tagged with green fluorescent protein (GFP) on theextracellular domain to facilitate the identification of transfectedcells. Such constructs were shown to preserve the properties of theglutamate receptor (Shi et al., 1999). NMDA receptor mediated currentwas recorded in the presence of 10 μM glycine. AMPA receptor current wasrecorded as described (Shi et al., 1999). Pipette solutions contained(in mM) Cs-methanesulfonate 110; CsCl 30; NaCl 4; HEPES 10; EGTA 5;CaCl₂ 0.5 adjusted to pH 7.3 with CsOH. 1 mM glutamate (for NMDAreceptor) or kainate (for AMPA receptor) was pressure (1.0 PSI) appliedthrough a puffer pipette positioned ˜10 μm to the cells. A program basedon Igor (WaveMetrics, Inc., Lake Oswego, Oreg.) was used to acquire dataon a Macintosh computer.

Rat Nucleic Acid and Polypeptide Sequences.

The following NR3B nucleic acid and amino acid sequences have beendetermined in rat: SEQ ID NOs: 5-11. SEQ ID NOs: 5 and 8 correspond toEST clone UI-R-B01-aiw-b-09-o-uI (Accession No: AW525909), SEQ ID NOs: 6and 9 correspond to EST clone UI-R-CAO-axg-c-09-0-UI (Accession No.:BE108608) and SEQ ID NOS: 7 and 10 correspond to UI-R-BJ2-avi-a-03-0-UI(Accession No: BE112464).

Results

Identification and Cloning of a New Mouse Glutamate Receptor

A unique sequence with significant homology to the glutamate receptorfamily (FIG. 1A) was identified by BLAST search of the published humanand mouse genomic sequences. Transcript levels and the tissuedistribution of mRNA were examined by RT-PCR of mouse RNA using a primerset spanning the predicted exons 1 and 2 (FIG. 2A). This revealed a bandof the expected size for spliced product in brainstem and spinal cord. Afainter band was also detected in the cerebellum. By use of other primersets covering exons 2 and 3, a similar pattern of expression wasdetected. More rostral structures did not show any significantexpression of this transcript, whereas RT-PCR for the NR1 subunitcarried out in parallel showed positive bands in all brain regions. Whenreverse transcriptase was omitted from the reaction (-RT), the band wasnot detected, ruling out amplification from contaminating genomic DNA. Afurther BLAST search identified EST clones from human, mouse, and rat.When the mouse EST clone AW045848 was used to probe polyA+RNA from mousespinal cord, it hybridized to a band at ˜3.5 kb (FIG. 2B). This clonewas used to screen a mouse spinal cord cDNA library. The screening ofapproximately 10⁶ clones yielded three positive clones, one of whichcontained full-length cDNA (accession AF396649).

Structure of-the NR3B gene, cDNA, and Protein Product

Sequence analysis of the full-length mouse clone revealed a cDNA of 3283bp encoding a polypeptide of 1003 amino acids (FIG. 1B). The cDNA wasfound to be encoded by at least nine exons in the mouse genome spanningapproximately 6.5 kb (FIG. 1D). A TATA box was found in the genomicsequence at −339 from the start codon ATG whereas the cDNA starts at−185, suggesting that there exists 5′-untranslated sequence beyond theCDNA. All nucleotides in the genomic sequence and the cDNA were found tobe identical, indicating that RNA editing does not occur in this gene.The predicted polypeptide has a significant similarity to NR3A (51%identity), and therefore it was named NR3B (FIG. 1A). A glutamatereceptor member named δ-2 has been reported in abstract form but due toa lack of detailed information, the identity of NR3B with δ-2 is unknown(Sevarino et al., 1996). The human sequence was assembled from thegenomic sequence deposited in GenBank (FIG. 1B). The C-terminus of thehuman clone loses homology after Gly890, which is followed by a possiblesplice donor site (GGGT), indicating there may be one or more additionalexon(s) in the human sequence. All the other exon-intron boundaries arepreserved between the two species and conform to a consensus splicedonor-acceptor site (GT-AG). The homology between the human and mouse is81.3%.

A full-length human NR3B CDNA sequence (SEQ ID NO: 3) that correspondsto the human NR3B sequence identified through homology to the mouse NR3Bsequence, is disclosed in PCT Application WO 01/44473. SEQ ID NO: 4 isthe polypeptide encoded by the human NR3B nucleic acid (SEQ ID NO: 3)and the polypeptide sequence (Accession no: AAC12680) corresponds to thehuman NR3B polypeptide identified through homology to the mouse NR3Bpolypeptide.

NR3B has a transmembrane topology typical of glutamate receptor with anN-terminal signal peptide and four membrane-associated regions. Five (inmouse) or four (in human) consensus sequences for N-glycosylation sitesare found on the N-terminal domain and one (for both species) at theloop between the third and fourth membrane-associated regions. Theintracellular C-terminus of mouse NR3B has three threonines and fiveserines, which may serve as regulatory phosphorylation sites. TheC-terminus ends with Ala-Glu-Ser, which does not conform to theconsensus PDZ domain protein-binding site sequence typical of otherglutamate receptor family members (Songyang et al., 1997; Sheng andSala, 2001).

The amino acid residues forming the glutamate-binding pocket have beenelucidated by structural studies of the crystallized ligand-bindingdomain of GluR2 (Armstrong and Gouaux, 2000). Importantly, the2-carboxyl group of glutamate binds to the guanidium group of Arg485 andthe NH group of Thr480 and the 2-amino group interacts with the carboxylgroup of Glu705 in GluR2. These residues can be mapped precisely on thesequence of NR3B with Thr480 of GluR2 corresponding to Ser533 of NR3B,Arg485 to Arg538, and Glu705 to Asp745. Other amino acids forming theputative ligand-binding pocket are also well conserved (FIG. 1B),indicating that NR3B itself is likely to bind to glutamate.

Expression of NR3B is Limited to Motoneurons

The RT-PCR revealed that NR3B is expressed selectively in a limitednumber of brain regions. Consistently, in situ hybridization of asagittal section of a mouse brain detected signal in limited regions inthe brainstem (FIG. 2C). A counterstaining of the sections showed thatthese are the trigeminal motor (V), facial (VII), and ambiguus nuclei(IX). A higher magnification of these structures revealed hybridizationsignals in motoneurons with large cell bodies (FIG. 2D). In contrast tothis, the signal was significantly weaker in motoneurons in the nucleicontrolling the external ocular muscles (oculomotor, III; trochlear, IV,abducens, VI, FIG. 2D).

In the spinal cord, the signal was also detected in the motoneurons inthe anterior horn (FIG. 3). However, in the L6 level, the signalintensity of NR3B in motoneurons was significantly weaker than higherlevels though the presence of motoneurons at this level was confirmed byNissl staining. These motoneurons were classified as those in Onuf'snucleus that controls external anal and urethral sphincters, based onplexus formation of serotonin immunoreactive fibers on cell body (FIG. 3bottom) (Micevych et al., 1986) and a comparison with thecytoarchitecture of the mouse spinal cord (Sidman et al., 1971) as wellas with the previous studies on rat counterpart.

NR3B acts as a Dominant-Negative Subunit

The electrophysiological properties of NR3B were tested by expressingNR3B in HEK293 cells. A conventional NMDA receptor channel is formed bya heteromeric combination of NR1, which is a key subunit forfunctionality, and at least one NR2 subunit, which is modulatory(Dingledine et al., 1999). NR3B cannot substitute for either of thesesubunits as expression of NR3B alone (n=6) or coexpression with NR1(n=6) or NR2A (n=5) did not result in electrophysiologically functionalchannels whereas coexpression of NR1 and NR2A gave a robust current(FIG. 4). However, coexpression of all three subunits markedly depressedthe whole-cell current compared with the combination of NR1 and NR2A(FIG. 4A, C). Increasing the amount of NR3B versus the other subunitshad a stronger suppressive effect (FIG. 4C). NR3B act as adominant-negative subunit and suppresses glutamate-induced whole-cellcurrent in cells coexpressing NR1 and NR2A. The distribution of averagedresponses obtained at +60 mV in Mg²⁺-free solution is shown in acumulative plot. (FIG. 4C). Increasing the amount of NR3B plasmid (0, 1,and 3 μg) versus other subunits (each 1 μg) caused a concomitantdecrease in current amplitude. Statistical significances were: 1:1:0 vs.1:1:1, p<0.05; 1:1:0 vs. 1:1:3, p<0.01; 1:1:1 vs. 1:1:3, p<0.01. AMPAreceptor mediated current was unchanged by the coexpression with NR3B.GluR2-(R586Q), a point mutation of the GluR2 subunit of AMPA receptorsthat gives a large current, was used because it was empirically knownthat this construct gives reliable response. This is not due to anon-specific reduction of the polypeptide expression level as a directquantification of expression levels of NR1 and NR2A tagged with GFPusing fluorescence as a measure did not show any significant differencein the presence or absence of NR3B. NR1 and NR2A tagged with GFP wereindividually expressed with NR3B and their expression levels weremeasured by the fluorescence intensity. The statistical significance inthis figure was assessed by Kolmogorov-Smirnov test. (FIG. 4D). Theeffect of NR3B is specific to NMDA receptor as the AMPA receptormediated current was not changed by the presence of NR3B (FIG. 4C). Thesensitivity of the remaining current to Mg²⁺ block was not affected bythe presence of NR3B subunit (FIG. 4B). The remaining current inNR3B-expressing cells exhibits a Mg²⁺ block indistinguishable from cellsnot expressing NR3B. The slight block at −60 mV and −80 mV may be due toresidual Mg²⁺ (FIG. 4B).

References

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The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, it being recognizedthat various modifications are possible within the scope of theinvention.

All references, publications and patents disclosed herein areincorporated by reference in their entirety.

1. An isolated nucleic acid molecule selected from the group consistingof: (a) nucleic acid molecules which hybridize under high stringencyconditions to a nucleic acid molecule having a nucleotide sequence setforth as SEQ ID NO: 1, providing that no more than about 18% of thenucleotides are changed from SEQ ID NO: 1, (b) nucleic acid moleculesthat differ from the nucleic acid molecules of (a) in codon sequence dueto the degeneracy of the genetic code, and (c) complements of (a) or(b), wherein the nucleic acid molecules or complements thereof code fora mouse NR3B NMDA receptor subunit.
 2. The isolated nucleic acidmolecule of claim 1, wherein the isolated nucleic acid moleculecomprises a nucleic acid sequence set forth as: SEQ ID NO:
 1. 3. Anisolated nucleic acid molecule selected from the group consisting of:(a) fragment of nucleotides 1-3290 of SEQ ID NO: 1 between 24 and 3289nucleotides in length, providing that no more than about 18% of thenucleotides are changed from SEQ ID NO:1, and (b) complements of (a). 4.An expression vector comprising the isolated nucleic acid molecule ofclaim 1 operably linked to a promoter.
 5. A host cell transformed ortransfected with the expression vector of claim
 4. 6. A transgenicnon-human animal comprising the expression vector of claim
 4. 7. Anisolated polypeptide encoded by the isolated nucleic acid molecule ofclaim
 1. 8. The isolated polypeptide of claim 7, wherein the isolatedpolypeptide comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO: 2 and a fragment or functional variant of SEQID NO:2.
 9. A binding polypeptide that selectively binds the isolatedpolypeptide of claim 7, wherein the binding polypeptide is an antibodyor antigen-binding fragment thereof.
 10. The binding polypeptide ofclaim 9, wherein the binding polypeptide selectively binds thepolypeptide sequence of SEQ ID NO:
 2. 11. A composition comprising amolecule selected from the group consisting of: (a) the nucleic acid ofclaim 1, (b) the polypeptide encoded by the isolated nucleic acidmolecule of claim 1, and (c) the binding polypeptide of claim 9, and apharmaceutically acceptable carrier.
 12. A method for making amedicament, comprising: placing in a pharmaceutically acceptablecarrier, a molecule selected from the group consisting of: (a) theisolated nucleic acid molecules of claim 1, (b) the isolated polypeptideof claim 7, and (c) the binding polypeptides of claim
 9. 13. The methodof claim 12, wherein the step of placing comprises placing atherapeutically effective amount of the molecule selected from the groupin the pharmaceutically acceptable carrier to form one or more doses.14. A method of making a glutamate receptor in vitro, comprisingintroducing glutamate receptor nucleic acids into a cell, wherein theglutamate receptor nucleic acids encode a NR3B polypeptide and whereinthe nucleic acids are as claimed in claim
 1. 15. The method of claim 14,wherein the NR3B subunit is encoded by the nucleotide sequence set forthas SEQ ID NO:
 1. 16. A method for diagnosing a motor neuron disordercharacterized by aberrant expression of a NR3B molecule, comprising:detecting expression of a NR3B molecule in a first biological sampleobtained from a subject, wherein a difference in expression level of theNR3B molecule compared to expression level a NR3B molecule in a controlsample indicates that the subject has a motor neuron disordercharacterized by aberrant expression of a NR3B molecule, and wherein theNR3B molecule is a nucleic acid molecule as claimed in claim 1 or is apolypeptide encoded by a nucleic acid molecule as claimed in claim 1.17. The method of claim 16, further comprising the steps of: detectingexpression of a NR3B molecule in a second biological sample obtainedfrom the subject at a time subsequent to the first biological sample,and comparing the expression of the NR3B molecule in the firstbiological sample and the second biological sample as an indication ofthe onset, progression, or regression of the motor neuron disorder. 18.The method of claim 17, wherein a decrease in expression level of theNR3B in the second biological sample compared to the expression level inthe NR3B in the first biological sample indicates progression of themotor neuron disorder characterized by aberrant expression of NR3B. 19.(canceled)
 20. The method of claim 17, wherein an increase in expressionlevel of the NR3B in the second biological sample compared to theexpression level in the NR3B in the first biological sample indicatesregression of the motor neuron disorder characterized by aberrantexpression of NR3B. 21-22. (canceled)
 23. The method of claim 16,comprising detecting expression of a NR3B nucleic acid molecule.
 24. Themethod of claim 23, wherein the NR3B nucleic acid molecule comprises thenucleic acid sequence set forth as SEQ ID NO: 1 or fragment thereof. 25.The method of claim 16, comprising detecting expression of a NR3Bpolypeptide.
 26. The method of claim 25, wherein the NR3B polypeptide isset forth as SEQ ID NO: 2 or fragment thereof.
 27. The method of claim16, wherein detecting comprises contacting the biological sample with anagent that selectively binds the NR3B molecule.
 28. The method of claim27, wherein the NR3B molecule is a nucleic acid and wherein the agentthat selectively binds the NR3B molecule is a nucleic acid selected fromthe group of nucleic acid molecules comprising the nucleotide sequencesthat hybridize to SEQ ID NO: 1 under high stringency conditions.
 29. Themethod of claim 27, wherein the NR3B molecule is a polypeptide andwherein the agent that selectively binds the NR3B molecule is a bindingpolypeptide selected from the group of binding polypeptides thatselectively bind to SEQ ID NO:
 2. 30. (canceled)
 31. A method forevaluating the effect of candidate pharmacological compounds onexpression of an NR3B subunit of a glutamate receptor, comprising:administering a candidate pharmaceutical agent to a subject; determiningthe effect of the candidate pharmaceutical agent on the expression levelof NR3B relative to the expression level of NR3B in a subject to whichno candidate pharmaceutical agent is administered, wherein a relativeincrease or relative decrease in the expression level of NR3B indicatesthe effect of the candidate pharmaceutical compound on the expression ofthe NR3B subunit of the glutamate receptor, and wherein NR3B is anucleic acid molecule as claimed in claim 1 or is a polypeptide encodedby a nucleic acid molecule as claimed in claim
 1. 32-33. (canceled) 34.A method for evaluating the effect of candidate pharmacologicalcompounds on the expression of a NR3B subunit of a glutamate receptor,comprising: contacting a candidate pharmaceutical agent with a NR3Bsubunit expressing cell or tissue sample; determining the effect of thecandidate pharmaceutical agent on the expression level of NR3B relativeto the expression level of NR3B in a NR3B subunit expressing cell ortissue sample not contacted with the candidate pharmaceutical agent,wherein a relative increase or relative decrease in the expression levelof NR3B indicates the effect of the candidate pharmaceutical compound onthe expression of NR3B subunit of a glutamate receptor and wherein NR3Bis a nucleic acid molecule as claimed in claim 1 or is a polypeptideencoded by a nucleic acid molecule as claimed in claim
 1. 35-36.(canceled)
 37. A method for diagnosing a motor neuron disordercharacterized by aberrant function of a NR3B molecule, comprising:detecting function of a NR3B molecule in a first biological sampleobtained from a subject, wherein a difference in function of the NR3Bmolecule compared to a NR3B molecule in a control sample indicates thatthe subject has a motor neuron disorder characterized by aberrantfunction of a NR3B molecule, and wherein the NR3B molecule is a nucleicacid molecule as claimed in claim 1 or is a polypeptide encoded by anucleic acid molecule as claimed in claim
 1. 38. The method of claim 37,further comprising the steps of: detecting function of a NR3B moleculein a second biological sample obtained from the subject at a timesubsequent to the first biological sample, and comparing the function ofthe NR3B molecule in the first biological sample and the secondbiological sample as an indication of the onset, progression, orregression of the motor neuron disorder.
 39. The method of claim 38,wherein a decrease in function level of the NR3B in the secondbiological sample compared to the function level in the NR3B in thefirst biological sample indicates progression of the motor neurondisorder characterized by aberrant function of NR3B.
 40. (canceled) 41.The method of claim 38, wherein an increase in function level of theNR3B in the second biological sample compared to the function level inthe NR3B in the first biological sample indicates regression of themotor neuron disorder characterized by aberrant expression of NR3B.42-43. (canceled)
 44. The method of claim 37, comprising detectingfunction of a NR3B nucleic acid molecule.
 45. The method of claim 44,wherein the NR3B nucleic acid molecule comprises a nucleic acid sequenceset forth as SEQ ID NO: 1 or fragment thereof.
 46. The method of claim37, comprising detecting function of a NR3B polypeptide.
 47. The methodof claim 46, wherein the NR3B polypeptide is set forth as SEQ ID NO: 2or fragment thereof.
 48. The method of claim 37, wherein detectingcomprises determining the cation passage through an NMDA receptorchannel.
 49. The method of claim 48, wherein the cation flux isdetermined with a method selected from the group consisting of:electrophysiological recording, drug screening assays, and ion-fluxmeasurement.
 50. (canceled)
 51. A method for evaluating the effect ofcandidate pharmacological compounds on function of an NR3B subunit of aglutamate receptor, comprising: administering a candidate pharmaceuticalagent to a subject that expresses a glutamate receptor containing afunctional NR3B subunit; detecting the function of the NR3B subunit ofthe glutamate receptor, determining the effect of the candidatepharmaceutical agent on the function level of NR3B relative to thefunction level of NR3B in a subject to which no candidate pharmaceuticalagent is administered, wherein a relative increase or relative decreasein the function level of NR3B indicates the effect of the candidatepharmacological compound on the function of the NR3B subunit of theglutamate receptor, and wherein NR3B is a nucleic acid molecule asclaimed in claim 1 or is a polypeptide encoded by a nucleic acidmolecule as claimed in claim
 1. 52-53. (canceled)
 54. The method ofclaim 51, wherein detecting comprises determining the cation passagethrough an NMDA receptor channel.
 55. The method of claim 51, whereinthe cation flux is determined with a method selected from the groupconsisting of: electrophysiological recording, drug screening assays,and ion-flux measurement.
 56. A method for evaluating the effect ofcandidate pharmacological compounds on function of a NR3B subunit of aglutamate receptor, comprising: contacting a glutamate receptor samplewith a candidate pharmaceutical agent; detecting the function of theNR3B subunit of the glutamate receptor, determining the effect of thecandidate pharmaceutical agent on the function level of NR3B relative tothe function level of NR3B in a glutamate receptor sample not contactedwith the candidate pharmaceutical agent, wherein a relative increase orrelative decrease in the function level of NR3B indicates the effect ofthe candidate pharmacological agent on the function of NR3B subunit ofthe glutamate receptors, and wherein NR3B is a nucleic acid molecule asclaimed in claim 1 or is a polypeptide encoded by a nucleic acidmolecule as claimed in claim
 1. 57-58. (canceled)
 59. The method ofclaim 56, wherein detecting comprises determining the cation passagethrough an NMDA receptor channel.
 60. The method of claim 56, whereinthe cation flux is determined with a method selected from the groupconsisting of: electrophysiological recording, drug screening assays,and ion-flux measurement.
 61. A kit for diagnosing a motor neurondisorder associated with aberrant expression of a NR3B molecule,comprising: one or more nucleic acid molecules that hybridize to a NR3Bnucleic acid molecule under high stringency conditions and instructionsfor the use of the nucleic acid molecules in the diagnosis of a motorneuron disorder associated with aberrant expression of a NR3B molecule,wherein the NR3B nucleic acid molecule is a nucleic acid molecule asclaimed in claim
 1. 62. The kit of claim 61, wherein the one or morenucleic acid molecules are a first primer and a second primer and,wherein the first primer and the second primer are constructed andarranged to selectively amplify at least a portion of an isolated NR3Bnucleic acid molecule comprising SEQ ID NO:
 1. 63. A kit for diagnosinga NR3B-associated motor neuron disorder in a subject comprising: one ormore binding polypeptides that selectively bind to a NR3B polypeptide,and instructions for the use of the binding polypeptides in thediagnosis of a motor neuron disorder associated with aberrant expressionof a NR3B molecule, wherein the NR3B polypeptide is encoded by a nucleicacid molecule as claimed in claim
 1. 64. (canceled)
 65. The kit of claim63, wherein the NR3B polypeptide is encoded by a nucleic acid comprisinga nucleotide sequence set forth as SEQ ID NO:
 1. 66. A method fortreating a subject with a motor neuron disorder characterized bydecreased expression of a NR3B molecule, comprising administering to thesubject an amount of a NR3B nucleic acid molecule effective to increaseexpression of a NR3B polypeptide and treat the motor neuron disorder,wherein the NR3B nucleic acid molecule is a nucleic acid molecule asclaimed in claim
 1. 67. A method for treating a subject with a motorneuron disorder characterized by decreased expression of a NR3Bpolypeptide, comprising administering to the subject an amount of a NR3Bpolypeptide effective to treat the motor neuron disorder, wherein theNR3B polypeptide is encoded by a nucleic acid molecule as claimed inclaim
 1. 68. A method for treating a subject with a motor neurondisorder characterized by increased expression of a NR3B nucleic acidmolecule, comprising administering to the subject an amount of anantisense molecule to a NR3B nucleic acid molecule effective to treatthe motor neuron disorder, wherein the NR3B nucleic acid molecule is anucleic acid molecule as claimed in claim
 1. 69. A method for treating asubject with a motor neuron disorder characterized by increasedexpression of a NR3B polypeptide, comprising: administering to thesubject an amount of a NR3B polypeptide binding polypeptide effective totreat the motor neuron disorder, wherein the NR3B polypeptide is apolypeptide encoded by a nucleic acid molecule as claimed in claim 1.70. (canceled)
 71. A method for treating a subject with a motor neurondisorder characterized by decreased function of a NR3B molecule,comprising administering to the subject an amount of a NR3B nucleic acidmolecule effective to treat the motor neuron disorder, wherein the NR3Bnucleic acid molecule is a nucleic acid molecule as claimed in claim 1.72. A method for treating a subject with a motor neuron disordercharacterized by decreased function of a NR3B polypeptide, comprisingadministering to the subject an amount of a NR3B polypeptide effectiveto treat the motor neuron disorder, wherein the NR3B polypeptide is apolypeptide encoded by a nucleic acid molecule as claimed in claim 1.73. A method for treating a subject with a motor neuron disordercharacterized by increased function of a NR3B nucleic acid molecule,comprising administering to the subject an amount of an antisensemolecule to a NR3B nucleic acid molecule effective to treat the motorneuron disorder, wherein the NR3B nucleic acid molecule is a nucleicacid molecule as claimed in claim
 1. 74. A method for treating a subjectwith a motor neuron disorder characterized by increased function of aNR3B polypeptide, comprising: administering to the subject an amount ofa NR3B polypeptide binding polypeptide effective to treat the motorneuron disorder, wherein the NR3B polypeptide is a polypeptide encodedby a nucleic acid molecule as claimed in claim
 1. 75. (canceled)
 76. Amethod for producing a NR3B polypeptide or fragment thereof, comprising:providing an isolated NR3B nucleic acid molecule operably linked to apromoter, wherein the NR3B nucleic acid molecule encodes the NR3Bpolypeptide or fragment thereof, and expressing the NR3B nucleic acidmolecule in an expression system, wherein the NR3B nucleic acid moleculeis a nucleic acid molecule as claimed in claim
 1. 77. The method ofclaim 76, further comprising: isolating the NR3B polypeptide or afragment thereof from the expression system.
 78. The method of claim 76,wherein the NR3B nucleic acid molecule is set forth as SEQ ID NO:
 1. 79.A method for making a NR3B polypeptide comprising: culturing the hostcell of claim 5, and isolating the NR3B polypeptide from the culture.80. A method for preparing a model of a motor neuron diseasecharacterized by aberrant expression of a NR3B molecule, comprisingintroducing into a cell, a NR3B molecule wherein the NR3B molecule is anucleic acid molecule as claimed in claim 1 or is a polypeptide encodedby a nucleic acid molecule as claimed in claim
 1. 81. (canceled)
 82. Themethod of claim 80, wherein the NR3B molecule is a NR3B nucleic acidmolecule set forth in SEQ ID NO:
 1. 83. The method of claim 80, whereinthe NR3B molecule is a NR3B polypeptide set forth in SEQ ID NO: 2.84-85. (canceled)
 86. A method for preparing an animal model of a motorneuron disorder characterized by aberrant function of a NR3B molecule,comprising: introducing into a non-human subject, an aberrant NR3Bmolecule; and detecting expression of the aberrant NR3B molecule in afirst biological sample obtained from the non-human subject, wherein theNR3B molecule is a nucleic acid molecule as claimed in claim 1 or is apolypeptide encoded by a nucleic acid molecule as claimed in claim 1.87-92. (canceled)
 93. A method for preparing a non-human animal model ofa motor neuron disorder characterized by reduced expression of a NR3Bmolecule, comprising administering to a non-human subject an effectiveamount of an anti-sense molecule to a NR3B nucleic acid molecule toreduce expression of the NR3B nucleic molecule in the non-human subject.94. The method of claim 93, wherein the NR3B molecule is a nucleic acidmolecule selected from the group containing SEQ ID NO: 1 and SEQ IDNO:3.
 95. A method for preparing a non-human animal model of a motorneuron disorder characterized by reduced expression of a NR3B molecule,comprising administering to a non-human subject an effective amount of abinding polypeptide to a NR3B polypeptide to reduce expression of theNR3B polypeptide in the non-human subject.
 96. (canceled)
 97. The methodof claim 95, wherein the NR3B molecule a nucleic acid molecule selectedfrom the group containing SEQ ID NO: 1 and SEQ ID NO:3.